Deuterated nucleoside prodrugs useful for treating hcv

ABSTRACT

Deuterated nucleoside analogs of Formula (I) and the pharmaceutically acceptable salts thereof are provided by this disclosure 
     
       
         
         
             
             
         
       
     
     The variables, e.g., B 1 , Y, R 1 , R 2 , R 3 , R 3 ′, R 4 , R 5 , R 6 , R 7 , R 8 , and R 9  carry definitions set forth in the disclosure. Compounds of Formula (I) are deuterated at one or more positions and the deuterium enrichment at each deuterated positions is at least 50%. Compounds and salts of Formula (I) are useful for treating viral infections, including HCV infections.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/811,464, filed Apr. 12, 2013, which is hereby incorporated byreference in its entirety.

BACKGROUND

An estimated 3% of the world's population is infected with the hepatitisC virus. Of those exposed to HCV, 80% to 85% become chronicallyinfected, at least 30% develop cirrhosis of the liver and 1-4% develophepatocellular carcinoma. Hepatitis C Virus (HCV) is one of the mostprevalent causes of chronic liver disease in the United States,reportedly accounting for about 15 percent of acute viral hepatitis, 60to 70 percent of chronic hepatitis, and up to 50 percent of cirrhosis,end-stage liver disease, and liver cancer. Chronic HCV infection is themost common cause of liver transplantation in the U.S., Australia, andmost of Europe. Hepatitis C causes an estimated 10,000 to 12,000 deathsannually in the United States. While the acute phase of HCV infection isusually associated with mild symptoms, some evidence suggests that onlyabout 15% to 20% of infected people will spontaneously clear HCV.

HCV is an enveloped, single-stranded RNA virus that contains apositive-stranded genome of about 9.6 kb. HCV is classified as a memberof the Hepacivirus genus of the family Flaviviridae. At least 4 strainsof HCV, GT-1-GT-4, have been characterized.

The HCV lifecycle includes entry into host cells; translation of the HCVgenome, polyprotein processing, and replicase complex assembly; RNAreplication, and virion assembly and release. In the RNA replicationprocess, a complementary negative strand copy of the genomic RNA isproduced. The negative strand copy is used as a template to synthesizeadditional positive strand genomic RNAs that may participate intranslation, replication, packaging, or any combination thereof toproduce progeny virus.

There are several proteins in hepatitis C that have been targeted fordrug therapy. NS5A is a zinc-binding proline rich hydrophilicphospho-protein with no inherent enzymatic activity, which can beinhibited with certain non-nucleotide compounds. NS5B is a key enzymewhich plays the major role in replicating HCV's viral RNA using a viralpositive RNA strand as a template, which has been inhibited withsynthetic nucleoside derivatives. NS2-3 protease is an enzymeresponsible for proteolytic cleavage between NS2 and NS3, which arenon-structural proteins. NS3 protease is responsible for the cleavage ofthe non-structural protein downstream. RNA helicase uses ATP hydrolysisto unwind RNA.

Sofosbuvir (Sovaldi, see structure below) is a nucleosidephosphoramidate NS5B inhibitor approved in December 2013 for thetreatment of HCV. The approved labeling recommends the followingregimens: (i) for genotypes 2 and 3 a 400 mg once a day oral tablet incombination with ribavirin and (ii) for genotypes 1 and 4 a 400 mg oncea day oral tablet (triple combination therapy) with ribavirin andpegylated interferon. The Sofosbuvir treatment lasts 12 weeks forgenotypes 1, 2 and 4 and 24 weeks for genotype 3. Sofosbuvir can also beused with ribavirin for the treatment of chronic hepatitis C patientswith hepatocellular carcinoma awaiting liver transplantation for up to48 weeks or until liver transplantation to prevent post-transplant HCVinfection. The FDA granted Sovaldi Priority Review and BreakthroughTherapy designation based on data from several large clinical trialsthat indicated a sustained viral response (SVR) of twelve weeks in 50-90percent of the trial participants. Patients who achieve “SVR12” areoften considered cured.

Alios BioPharma, Inc. licensed ALS-2200 to Vertex Pharmaceuticals Inc.for hepatitis C treatment development in June 2011. ALS-2200 is amixture of diastereomers at a chiral phosphorus stereocenter. Vertexchanged the name to VX-135, which is currently in Phase II clinicaltrials. While the companies have not disclosed the chemical structure ofVX-135, they have said that it is a nucleotide analog prodrug, and anNS5B inhibitor. In 2013, the FDA placed VX-135 on partial clinical holdafter three patients receiving high dosages of the medicine showed livertoxicity. Lowering the dose of a nucleoside inhibitor to avoid toxicitycan sometimes also compromise or lower efficacy. Vertex announced inJanuary 2014 that VX-135 in combination with daclastavir (Bristol-MyersSquibb NS5A inhibitor) had completed a Phase 2a trial. In anintent-to-treat analysis, the sustained viral response rate four weeksafter the completion of treatment (SVR4) was 83% (10 of 12) intreatment-naïve genotype 1 infected individuals who received 200 mgVX-135 in combination with daclatasvir. One patient exhibited a seriousadverse event of vomiting/nausea. The eleven remaining patientscompleted 12 weeks of treatment, for a completion of treatment rate(SVR4) of 91%.

Idenix Pharmaceuticals Inc. is developing IDX21437 for the treatment ofhepatitis C, which is a uridine nucleoside prodrug NS5B inhibitor. Thedetails of the chemical structure have not been released to date. InApril 2014, Idenix announced that once-daily 300 mg IDX21437 for sevendays led to a mean maximum reduction in viral load of 4.2-4.3 log 10IU/mL in 18 treatment naïve patients with genotype 1, 2 or 3.

Despite progress in the area of hepatitis C treatment, there have alsobeen a number of difficult setbacks. BMS-986094, a guanine-basedphosphoramidate for hepatitis C was pulled from clinical trials afterthe death of a patient due to heart failure. BMS thereafter announced in2013 that it was exiting the hepatitis C research area. Following theBMS drug withdrawal, Idenix Pharmaceuticals's similar phosphoramidateNS5B inhibitor, IDX 19368, was placed on clinical hold and ultimatelydiscontinued. This followed the previous clinical hold anddiscontinuation of development of the phosphoramidate IDX184 for thesame indication.

It is known that effective treatment against hepatitis C includescombination therapy, due to the onset of viral resistance duringmonotherapy. Given the documented challenges of developing optimalhepatitis C agents, and the fact that multiple optimal agents arerequired for effective therapy, there is a strong need for additionalhepatitis C agents.

SUMMARY

Nucleoside analogs are a class of compounds that have known anti-viralactivity, including, in some instances, anti-HCV activity. Nucleosideanalogs are particularly useful in combination with other direct-actinganti-HCV compounds.

The present disclosure provides a novel class of deuterated nucleosideanalogs of Formula (I) and the pharmaceutically acceptable salts thereof

Within Formula (I) the variables, e.g., B₁, Y, A, R₁, R₂, R₃, R₃′, R₄,R₅, R₆, R₇, R₈, and R₉ carry the following definitions.

Y is NH or O and Z is CH₂ or O.

R₁ is hydroxyl, fluoro, or —OCD₃ and R₂ is hydrogen or deuterium or R₂is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, or C₂-C₆alkynyl; each of which isoptionally deuterated and optionally substituted.

Or, R₁ and R₂ are joined to form a 3- to 6-membered cycloalkyl ring or a3- to 6-membered heterocycloalkyl ring containing one heteroatomselected from N, O, and S, each of which is optionally substituted.

R₃ is hydrogen, deuterium, halogen, or —N₃; or R₃ is C₁-C₆alkyl,allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, (C₃-C₆cycloalkyl)C₀-C₄carbhydryl,(4- to 6-membered heterocycloalkyl)C₀-C₄carbhydryl,(aryl)C₀-C₄carbhydryl, or (heteroaryl)C₀-C₄carbhydryl, each of which isoptionally deuterated and optionally substituted; and R₃′ is hydroxyl.

Or, R₃ and R₃′ are taken together to form a 3- to 6-membered ringoptionally containing one heteroatom selected from N, O, and S, whichring is optionally substituted with one or more substituentsindependently selected from halogen, hydroxyl, C₁-C₂alkyl, andC₁-C₂alkoxy.

R₄ is hydrogen, deuterium, halogen, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy;or R₄ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, orC₁-C₆alkoxy, each of which is optionally deuterated and optionallysubstituted; and R₅ is hydrogen, deuterium, or halogen; or R₅ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or C₁-C₆alkoxy, each ofwhich is optionally deuterated and optionally substituted.

Or, R₄ and R₅ are taken together to form a 3- to 6-membered ringoptionally containing one heteroatom selected from N, O, and S, whichring is optionally substituted with one or more substituentsindependently selected from halogen, hydroxyl, C₁-C₂alkyl, andC₁-C₂alkoxy.

R₆ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, (aryl)C₀-C₂alkyl,or 5- to 6-membered monocyclic heteroaryl containing 1 to 3 heteroatomsindependently chosen from N, O, and S, or 8- to 10-membered bicyclicheteroaryl containing 1 to 4 heteroatoms independently chosen from N, 0,and S; each of which R₆ is optionally substituted.

R₇ is hydrogen, halogen, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy; or R₇ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆alkoxy,(C₃-C₆cycloalkyl)C₀-C₄alkyl, or (aryl)C₀-C₂alkyl; each of which isoptionally substituted; and R₈ is hydrogen, halogen, C₁-C₂haloalkyl, orC₁-C₂haloalkoxy; or R₈ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, or C₁-C₆alkoxy, each of which is optionally substituted.

Or, R₇ and R₈ are taken together to form a 3- to 6-membered cycloalkylring or 3- to 6-membered heterocycloalkyl ring containing one heteroatomchosen from N, O, and S; each of which is optionally substituted.

R₉ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl,(C₃-C₇cycloalkyl)C₀-C₄carbhydryl, (aryl)C₀-C₄carbhydryl, (3- to6-membered heterocycloalkyl)C₀-C₄carbhydryl, or(heteroaryl)C₀-C₄carbhydryl, each of which is optionally substituted.

B₁ is a base selected from

R₁₀ and R₁₁ are independently hydrogen and deuterium.

R₁₂, R₁₃, and R₁₃′ are independently hydrogen, deuterium, methyl, and—CD₃.

R₁₄ is hydrogen, deuterium, hydroxyl, amino; C₁-C₄alkoxy, deuteratedC₁-C₄alkoxy, C₁-C₄alkylester, or mono- or di-C₁-C₄alkylcarbamate.

Wherein each position represented by D has a deuterium enrichment of atleast 50%; and one or more of R₁, R₂, R₄, R₅, R₁₀, R₁₁, R₁₂, R₁₃, R₁₃′,and R₁₄ is deuterium with a deuterium enrichment of at least 50% or adeuterated group with at least one position having a deuteriumenrichment of at least 50%. The disclosure also includes apharmaceutical composition comprising a compound or salt of Formula (I)together with a pharmaceutically acceptable carrier. The pharmaceuticalcomposition may contain a compound or salt of Formula (I) as the onlyactive agent or may contain one or more additional active agents, suchas an HCV HS3 protease inhibitor and an HCV NS5a inhibitor.

The disclosure also includes a method of treating hepatitis C infection,comprising providing a therapeutically effective amount of a compound ofFormula (I) or a pharmaceutical composition containing a compound ofFormula (I) to a patient in need thereof.

DETAILED DESCRIPTION Chemical Description and Terminology

Compounds are described using standard nomenclature. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs. Unless clearly contraindicated by the contexteach compound name includes the free acid or free base form of thecompound as well as all pharmaceutically acceptable salts of thecompound.

The term “Formula (I)” encompasses all compounds that satisfy Formula(I), including any enantiomers, racemates and stereoisomers, as well asall pharmaceutically acceptable salts of such compounds. “Formula (I)”includes all subgeneric groups of Formula (I), such as Formula (II).Formula (III), and Formula (IV) and also includes pharmaceuticallyacceptable salts of a compound of Formula (I), unless clearlycontraindicated by the context in which this phrase is used.

The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item. Theterm “or” means “and/or”. The open-ended transitional phrase“comprising” encompasses the intermediate transitional phrase“consisting essentially of” and the close-ended phrase “consisting of”Claims reciting one of these three transitional phrases, or with analternate transitional phrase such as “containing” or “including” can bewritten with any other transitional phrase unless clearly precluded bythe context or art. Recitation of ranges of values are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. The endpoints of all rangesare included within the range and independently combinable. All methodsdescribed herein can be performed in a suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”), isintended merely to better illustrate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention asused herein. Unless defined otherwise, technical and scientific termsused herein have the same meaning as is commonly understood by one ofskill in the art to which this invention belongs.

Compounds of Formula (I) include compounds of the formula havingisotopic substitutions at any position. Isotopes include those atomshaving the same atomic number but different mass numbers. By way ofgeneral example, and without limitation, isotopes of hydrogen includetritium and deuterium and isotopes of carbon include ¹¹C, ¹³C, and ¹⁴C.While the compounds of Formula (I) require a moderate or high enrichmentof deuteration (substitution of a hydrogen with deuterium) at identifiedpositions, Formula (I) includes embodiments in which other positions areisotopically enriched.

An “active agent” means a compound (including a compound disclosedherein), element, or mixture that when administered to a patient, aloneor in combination with another compound, element, or mixture, confers,directly or indirectly, a physiological effect on the patient. Theindirect physiological effect may occur via a metabolite or otherindirect mechanism.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —(C═O)NH₂is attached through carbon of the keto (C═O) group.

“Alkyl” is a branched or straight chain saturated aliphatic hydrocarbongroup, having the specified number of carbon atoms, generally from 1 toabout 12 carbon atoms. The term C₁-C₆alkyl as used herein indicates analkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Otherembodiments include alkyl groups having from 1 to 8 carbon atoms, 1 to 4carbon atoms or 1 or 2 carbon atoms, e.g. C₁-C₈alkyl, C₁-C₄alkyl, andC₁-C₂alkyl. When C₀-C_(n) alkyl is used herein in conjunction withanother group, for example, (C₃-C₇cycloalkyl)C₀-C₄ alkyl, the indicatedgroup, in this case cycloalkyl, is either directly bound by a singlecovalent bond (C₀alkyl), or attached by an alkyl chain having thespecified number of carbon atoms, in this case 1, 2, 3, or 4 carbonatoms. Examples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, andsec-pentyl.

“Alkenyl” is a branched or straight chain aliphatic hydrocarbon grouphaving one or more double carbon-carbon bonds that may occur at anystable point along the chain, having the specified number of carbonatoms. Examples of alkenyl include, but are not limited to, ethenyl andpropenyl.

“Alkynyl” is a branched or straight chain aliphatic hydrocarbon grouphaving one or more triple carbon-carbon bonds that may occur at anystable point along the chain, having the specified number of carbonatoms. Examples of alkynyl include, but are not limited to, ethynyl andpropynyl.

“Allenyl” is an alkenyl group having two consecutive double bonds, i.e.,a group of formula —C═C═CH₂.

“Alkoxy” is an alkyl group as defined above with the indicated number ofcarbon atoms covalently bound to the group it substitutes by an oxygenbridge (—O—). Examples of alkoxy include, but are not limited to,methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy,n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy,2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

“Alkanoyl” is an alkyl group as defined above with the indicated numberof carbon atoms covalently bound to the group is substitutes through acarbonyl (C═O) bridge. The carbonyl carbon is included in the number ofcarbons, that is C₂alkanoyl is a CH₃(C═O)— group.

“Alkylester” is an alkyl group as defined herein covalently bound to thegroup it substitutes by an ester linkage. The ester linkage may be ineither orientation, e.g., a group of the formula —O(C═O)alkyl or a groupof the formula —(C═O)Oalkyl.

“Cycloalkyl” is a saturated hydrocarbon ring group, having the specifiednumber of carbon atoms. Monocyclic cycloalkyl groups typically have from3 to about 8 carbon ring atoms or from 3 to 7 (3, 4, 5, 6, or 7) carbonring atoms. Cycloalkyl substituents may be pendant from a substitutednitrogen or carbon atom, or a substituted carbon atom that may have twosubstituents may have a cycloalkyl group, which is attached as a spirogroup. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl.

“Haloalkyl” indicates both branched and straight-chain alkyl groupshaving the specified number of carbon atoms, substituted with 1 or morehalogen atoms, up to the maximum allowable number of halogen atoms.Examples of haloalkyl include, but are not limited to, trifluoromethyl,difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.

“Haloalkoxy” indicates a haloalkyl group as defined herein attachedthrough an oxygen bridge (oxygen of an alcohol radical).

“Halo” or “halogen” indicates any of fluoro, chloro, bromo, and iodo.

“Aryl” indicates aromatic groups containing only carbon in the aromaticring or rings. Typical aryl groups contain 1 to 3 separate, fused, orpendant rings and from 6 to about 18 ring atoms, without heteroatoms asring members. When indicated, such aryl groups may be furthersubstituted with carbon or non-carbon atoms or groups. Such substitutionmay include fusion to a 5 to 7-membered saturated cyclic group thatoptionally contains 1 or 2 heteroatoms independently chosen from N, O,and S, to form, for example, a 3,4-methylenedioxy-phenyl group. Arylgroups include, for example, phenyl, naphthyl, including 1-naphthyl and2-naphthyl, and bi-phenyl.

“Heteroaryl” indicates a stable monocyclic aromatic ring having theindicated number of ring atoms which contains from 1 to 3, or in someembodiments from 1 to 2, heteroatoms chosen from N, O, and S, withremaining ring atoms being carbon, or a stable bicyclic or tricyclicsystem containing at least one 5- to 7-membered aromatic ring whichcontains from 1 to 3, or in some embodiments from 1 to 2, heteroatomschosen from N, O, and S, with remaining ring atoms being carbon.Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms. Insome embodiments bicyclic heteroaryl groups are 9- to 10-memberedheteroaryl groups, that is, groups containing 9 or 10 ring atoms inwhich one 5- to 7-member aromatic ring is fused to a second aromatic ornon-aromatic ring. When the total number of S and O atoms in theheteroaryl group exceeds 1, these heteroatoms are not adjacent to oneanother. It is preferred that the total number of S and O atoms in theheteroaryl group is not more than 2. It is particularly preferred thatthe total number of S and O atoms in the aromatic heterocycle is notmore than 1. Examples of heteroaryl groups include, but are not limitedto, oxazolyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl,pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl,thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzo[d]oxazolyl,benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl,dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, and isoxazolyl.“Heteroaryloxy” is a heteroaryl group as described bound to the group itsubstituted via an oxygen bridge.

“Heterocycloalkyl” is a saturated ring group, having 1, 2, 3, or 4heteroatoms independently chosen from N, S, and O, with remaining ringatoms being carbon. Monocyclic heterocycloalkyl groups typically havefrom 3 to about 8 ring atoms or from 4 to 6 ring atoms. Examples ofheterocycloalkyl groups include morpholinyl, piperazinyl, piperidinyl,and pyrrolinyl.

“Carbhydryl” is a saturated or unsaturated aliphatic group containingthe indicated number of carbon atoms. “carbhydryl” may be used inconjunction with other groups, such as aryl, as in “(aryl)carbhydryl.”

The term “mono- and/or di-alkylamino” indicates secondary or tertiaryalkyl amino groups, wherein the alkyl groups are independently chosenalkyl groups, as defined herein, having the indicated number of carbonatoms. The point of attachment of the alkylamino group is on thenitrogen. Examples of mono- and di-alkylamino groups include ethylamino,dimethylamino, and methyl-propyl-amino.

“Mono- and/or di-alkylcarbamate” includes mono-alkylcarbamate groups offormula (alkyl₁)O(C═O)NH— or a dialkylcarboxamide groups of the formula(alkyl₁)O(C═O)N(alkyl₂)—in which the point of attachment of the mono- ordialkylcarboxamide substituent to the molecule it substitutes is on thenitrogen of the carbamate amino. The term “mono and/ordi-alkylcarbamate” also includes groups of the formula (alkyl₁)NH(C═O)O—and (alkyl₁)N(alkyl₂)(C═O)O— in which the carbamate is covalently boundto the group it substitutes by its non-keto oxygen atom. The groupsalkyl₁ and alkyl₂ are independently chosen alkyl groups, carrying thealkyl definition set forth in this disclosure and having the indicatednumber of carbon atoms.

The term “substituted”, as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a selectionfrom the indicated group, provided that the designated atom's normalvalence is not exceeded. When the substituent is oxo (i.e., ═O) then 2hydrogens on the atom are replaced. When an oxo group substitutesaromatic moieties, the corresponding partially unsaturated ring replacesthe aromatic ring. For example a pyridyl group substituted by oxo is apyridone. Combinations of substituents and/or variables are permissibleonly if such combinations result in stable compounds or useful syntheticintermediates. A stable compound or stable structure is meant to imply acompound that is sufficiently robust to survive isolation from areaction mixture, and subsequent formulation into an effectivetherapeutic agent. Unless otherwise specified substituents are namedinto the core structure. For example, it is to be understood that whenaminoalkyl is listed as a possible substituent the point of attachmentof this substituent to the core structure is in the alkyl portion.

Suitable groups that may be present on a “substituted” or “optionallysubstituted” position include, but are not limited to, e.g., halogen;cyano; hydroxyl; nitro; azido; alkanoyl (such as a C₂-C₆ alkanoylgroup); carboxamide; alkyl groups (including cycloalkyl groups) having 1to about 8 carbon atoms, or 1 to about 6 carbon atoms; alkenyl andalkynyl groups including groups having one or more unsaturated linkagesand from 2 to about 8, or 2 to about 6 carbon atoms; alkoxy groupshaving one or more oxygen linkages and from 1 to about 8, or from 1 toabout 6 carbon atoms; aryloxy such as phenoxy; alkylthio groupsincluding those having one or more thioether linkages and from 1 toabout 8 carbon atoms, or from 1 to about 6 carbon atoms; alkylsulfinylgroups including those having one or more sulfinyl linkages and from 1to about 8 carbon atoms, or from 1 to about 6 carbon atoms;alkylsulfonyl groups including those having one or more sulfonyllinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; aminoalkyl groups including groups having one or more N atoms andfrom 1 to about 8, or from 1 to about 6 carbon atoms; aryl having 6 ormore carbons and one or more rings, (e.g., phenyl, biphenyl, naphthyl,or the like, each ring either substituted or unsubstituted aromatic);arylalkyl having 1 to 3 separate or fused rings and from 6 to about 18ring carbon atoms, with benzyl being an exemplary arylalkyl group;arylalkoxy having 1 to 3 separate or fused rings and from 6 to about 18ring carbon atoms, with benzyloxy being an exemplary arylalkoxy group;or a saturated, unsaturated, or aromatic heterocyclic group having 1 to3 separate or fused rings with 3 to about 8 members per ring and one ormore N, O or S atoms, e.g. coumarinyl, quinolinyl, isoquinolinyl,quinazolinyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, pyrrolyl,thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl, imidazolyl,indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, andpyrrolidinyl. Such heterocyclic groups may be further substituted, e.g.with hydroxy, alkyl, alkoxy, halogen and amino. In certain embodiments“optionally substituted” includes one or more substituents independentlychosen from halogen, hydroxyl, amino, cyano, —CHO, —COOH, —CONH₂,C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆alkoxy, C₂-C₆alkanoyl, C₁-C₆alkylester,(mono- and di-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

Deuteration” and “deuterated” mean that a hydrogen at the specifiedposition is replaced by deuterium. In any sample of a compound ofFormula I in which a position is deuterated some discrete molecules ofthe compound of Formula I will likely have hydrogen, rather thandeuterium, at the specified position. However the percent of moleculesof the compound of Formula I in the sample which have deuterium at thespecified position will be much greater than would naturally occur. Thedeuterium at the deuterated position is enriched. The term “enriched” asused herein, refers to the percentage of deuterium versus other hydrogenspecies at that location. As an example, if it is said that a positionin the compound of Formula I contains 50% deuterium enrichment, thatmeans that rather than hydrogen at the specified position the deuteriumcontent is 50%. For clarity, it is confirmed that the term “enriched” asused herein does not mean percentage enriched over natural abundance. Inone embodiment, deuterated compounds of Formula I will have at least 10%deuterium enrichment at any deuterated position. In other embodiments,there will be at least 50%, at least 90%, or at least 95% deuteriumenrichment at the specified deuterated position or positions. A“deuterated substituent” is a substituent in which at least one hydrogenis replaced by deuterium at the specified percent enrichment.“Optionally deuterated” means that the position may be either hydrogenand the amount of deuterium at the position is only the naturallyoccurring level of deuterium or the position is enriched with deuteriumabove the naturally occurring deuterium level.

A “dosage form” means a unit of administration of an active agent.Examples of dosage forms include tablets, capsules, injections,suspensions, liquids, emulsions, creams, ointments, suppositories,inhalable forms, transdermal forms, and the like.

“Pharmaceutical compositions” are compositions comprising at least oneactive agent, such as a compound or salt of Formula (I), and at leastone other substance, such as a carrier. Pharmaceutical compositionsoptional contain one or more additional active agents. When specified,pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturingpractice) standards for human or non-human drugs. “Pharmaceuticalcombinations” are combinations of at least two active agents which maybe combined in a single dosage form or provided together in separatedosage forms with instructions that the active agents are to be usedtogether to treat a disorder, such as hepatitis C.

“Pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds in which the parent compound is modified by makinginorganic and organic, non-toxic, acid or base addition salts thereof.The salts of the present compounds can be synthesized from a parentcompound that contains a basic or acidic moiety by conventional chemicalmethods. Generally, such salts can be prepared by reacting free acidforms of these compounds with a stoichiometric amount of the appropriatebase (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or thelike), or by reacting free base forms of these compounds with astoichiometric amount of the appropriate acid. Such reactions aretypically carried out in water or in an organic solvent, or in a mixtureof the two. Generally, non-aqueous media like ether, ethyl acetate,ethanol, isopropanol, or acetonitrile are preferred, where practicable.Salts of the present compounds further include solvates of the compoundsand of the compound salts.

Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts and the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, conventional non-toxic acid salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Lists of additionalsuitable salts may be found, e.g., in Remington Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

The term “carrier” applied to pharmaceutical compositions/combinationsof the invention refers to a diluent, excipient, or vehicle with whichan active compound is provided.

A “pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition/combination that isgenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes an excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the present application includes both one and morethan one such excipient.

A “patient” is a human or non-human animal in need of medical treatment.Medical treatment can include treatment of an existing condition, suchas a disease or disorder, prophylactic or preventative treatment, ordiagnostic treatment. In some embodiments the patient is a humanpatient.

“Providing” means giving, administering, selling, distributing,transferring (for profit or not), manufacturing, compounding, ordispensing.

“Providing a compound of Formula (I) with at least one additional activeagent” means the compound of Formula (I) and the additional activeagent(s) are provided simultaneously in a single dosage form, providedconcomitantly in separate dosage forms, or provided in separate dosageforms for administration separated by some amount of time that is withinthe time in which both the compound of Formula (I) and the at least oneadditional active agent are within the blood stream of a patient. Incertain embodiments the compound of Formula (I) and the additionalactive agent need not be prescribed for a patient by the same medicalcare worker. In certain embodiments the additional active agent oragents need not require a prescription. Administration of the compoundof Formula (I) or the at least one additional active agent can occur viaany appropriate route, for example, oral tablets, oral capsules, oralliquids, inhalation, injection, suppositories or topical contact.

“Treatment,” as used herein includes providing a compound of Formula(I), either as the only active agent or together with at least oneadditional active agent sufficient to: (a) prevent a disease or asymptom of a disease from occurring in a patient who may be predisposedto the disease but has not yet been diagnosed as having it (e.g.including diseases that may be associated with or caused by a primarydisease (as in liver fibrosis that can result in the context of chronicHCV infection); (b) inhibiting the disease, i.e. arresting itsdevelopment; and (c) relieving the disease, i.e., causing regression ofthe disease. “Treating” and “treatment” also means providing atherapeutically effective amount of a compound of Formula (I), as theonly active agent or together with at least one additional active agentto a patient having or susceptible to a hepatitis C infection.

A “therapeutically effective amount” of a pharmaceuticalcomposition/combination of this invention means an amount effective,when administered to a patient, to provide a therapeutic benefit such asan amelioration of symptoms, e.g., an amount effective to decrease thesymptoms of a hepatitis C infection. For example a patient infected witha hepatitis C virus may present elevated levels of certain liverenzymes, including AST and ALT. A therapeutically effect amount is thusan amount sufficient to provide a significant reduction in elevated ASTand ALT levels or an amount sufficient to provide a return of AST andALT levels to the normal range. A therapeutically effective amount isalso an amount sufficient to prevent a significant increase orsignificantly reduce the detectable level of virus or viral antibodiesin the patient's blood, serum, or tissues. One method of determiningtreatment efficacy includes measuring HCV RNA levels by a conventionalmethod for determining viral RNA levels such as the Roche TaqMan assay.In certain preferred embodiments treatment reduces HCV RNA levels belowthe limit of quantitation (30 IU/mL, as measured by the Roche TaqMan(R)assay) or more preferably below the limit of detection (10 IU/mL, RocheTaqMan).

A significant increase or reduction in the detectable level of virus orviral antibodies is any detectable change that is statisticallysignificant in a standard parametric test of statistical significancesuch as Student's T-test, where p<0.05.

Chemical Description

Formula (I) includes all subformulae thereof. In certain situations, thecompounds of Formula (I) may contain one or more asymmetric elementssuch as stereogenic centers, stereogenic axes and the like, e.g.asymmetric carbon atoms, so that the compounds can exist in differentstereoisomeric forms. These compounds can be, for example, racemates oroptically active forms. For compounds with two or more asymmetricelements, these compounds can additionally be mixtures of diastereomers.For compounds having asymmetric centers, it should be understood thatall of the optical isomers and mixtures thereof are encompassed, unlessthe stereochemistry is explicitly stated. In addition, compounds withcarbon-carbon double bonds may occur in Z- and E-forms, with allisomeric forms of the compounds being included in the presentdisclosure. In these situations, single enantiomers, i.e., opticallyactive forms, can be obtained by asymmetric synthesis, synthesis fromoptically pure precursors, or by resolution of the racemates. Resolutionof the racemates can also be accomplished, for example, by conventionalmethods such as crystallization in the presence of a resolving agent, orchromatography, using, for example using a chiral HPLC column.

Where a compound exists in various tautomeric forms, the invention isnot limited to any one of the specific tautomers, but rather includesall tautomeric forms.

Certain compounds are described herein using a general formula thatincludes variables, e.g. B₁, R₁-R₉. Unless otherwise specified, eachvariable within such a Formula (I) is defined independently of othervariables. Thus, if a group is said to be substituted, e.g. with 0-2 R*,then the group may be substituted with up to two R* groups and R* ateach occurrence is selected independently from the definition of R*.Also, combinations of substituents and/or variables are permissible onlyif such combinations result in stable compounds.

In addition the compounds and salts of Formula (I) discussed in theSUMMARY section, the disclosure includes compounds and salt of Formula(I), (II) and (III)

In another embodiment, the nucleoside derivative of Formula (I), (II) or(III) is administered as a phosphorus R or S stereoisomer, which is atleast in 90% pure form, and typically, 95, 98 or 99% pure form. Inanother embodiment the compounds or salt of Formula (I), (II), or (III)is supplied as a 50/50 mixture of stereoisomers at the phosphorus chiralcenter.

Deuteration

The Each position represented by D in Formula (I)-(III) has a deuteriumenrichment of at least 50%. One or more of R₁, R₂, R₄, R₅, R₁₀, R₁₁,R₁₂, R₁₃, R₁₃′, and R₁₄ in the compound of Formula (I)-(III) isdeuterium with a deuterium enrichment of at least 50% or a deuteratedsubstituent with at least one position in the deuterated substituenthaving a deuterium enrichment of at least 50%.

Each position represented by D in Formula (I)-(III) has a deuteriumenrichment of at least 90%. One or more of R₁, R₂, R₄, R₅, R₁₀, R₁₁,R₁₂, R₁₃, R₁₃′, and R₁₄ in the compound of Formula (I)-(III)) isdeuterium with a deuterium enrichment of at least 90% or a deuteratedsubstituent with at least one position in the deuterated substituenthaving a deuterium enrichment of at least 90%.

Each position represented by D in Formula (I)-(III) has a deuteriumenrichment of at least 90%. At least 2 of R₁, R₂, R₄, R₅, R₁₀, R₁₁, R₁₂,R₁₃, R₁₃′, and R₁₄ are deuterium with a deuterium enrichment of at least90% or a deuterated substituent with at least one position in thedeuterated substituent having a deuterium enrichment of at least 90%.Each position represented by D in Formula (I)-(III) has a deuteriumenrichment of at least 95%. 2 or 3 of R₁, R₂, R₄, R₅, R₁₀, R₁₁, R₁₂,R₁₃, R₁₃′, and R₁₄ are deuterium with a deuterium enrichment of at least95% or a deuterated substituent with at least one position in thedeuterated substituent having a deuterium enrichment of at least 95%.

Each position represented by D in Formula (I)-(III) has a deuteriumenrichment of at least 90%. 3 of R1, R₂, R₄, R₅, R₁₀, R₁₁, R₁₂, R₁₃,R₁₃′, and R₁₄ are deuterium with a deuterium enrichment of at least 90%or a deuterated substituent with at least one position in the deuteratedsubstituent having a deuterium enrichment of at least 90%.

Each position represented by D in Formula (I)-(III) has a deuteriumenrichment of at least 90%. At least R₄, R₅ and one of R₁₂ and R₁₃ aredeuterium with a deuterium enrichment of at least 90% or a deuteratedsubstituent with at least one position in the deuterated substituenthaving a deuterium enrichment of at least 90%.

It has been surprisingly discovered that compounds of Formula (I)-(III)in which R₄ and R₅ are both deuterium with a deuterium enrichment of atleast 90% are particularly effective NS5B inhibitors and thus useful forthe treatment of hepatitis C. In certain embodiments it is alsopreferred that R₂ is methyl and R₁ is hydroxyl.

Upon administration to a patient a compound of Formula (I), (II), or(III) is first converted to the nucleoside monophosphate which is thenfurther phosphorylated to the nucleoside triphosphate, which is theactive species. Alternatively the nucleoside monophosphate may bedephosphorylated to the free nucleoside. The free nucleoside is inactiveas a NS5B inhibitor. Nucleoside triphosphate (NTP) is the active speciesthat inhibits viral replication in hepatocytes and its levels andintrinsic potency drive the effectiveness of the treatment.

It has surprisingly been discovered that deuteration of the 5′-positionof the nucleoside stabilizes the nucleoside derivative fromdephosphorylation to the undesired 5′-OH, 5′-deuterated-nucleoside. Thisis surprising because the deuterium atom(s) are not cleaved duringdephosphorylation and are not bound to an atom that is cleaved duringdephosphorylation. The disclosure includes the use 5′-deuterium toproduce a significant effect on metabolism and efficacy through a remoteand unexpectedly important secondary deuterium isotope effect. Such animportant secondary deuterium isotope effect on de-monophosphorylationat the 5′-position has not been previously reported. By increasing thestability of the 5′-monophosphate of the nucleoside againstdephosphorylation, an increase in the active 5′-triphosphate pool of thenucleoside is achieved, which can result in increased efficacy at agiven oral dosage or equal efficacy using a lower dose of thenucleoside. It may also have a significant effect on the half-life, andthus pharmacokinetics, of the drug.

Therefore, in another embodiment, the present disclosure includes amethod for treating a host afflicted with a disorder that is treatablewith a nucleoside or nucleotide, the improvement comprising substitutingone or both of the hydrogens at the 5′-position of the nucleoside ornucleotide with a deuterium with at least 90% enrichment over protium(i.e., less than 10% ¹H hydrogen) (and in other embodiments, 50, 95, 98or 99% enrichment). The therapeutic effect of any nucleotide ornucleoside can be enhanced if the active metabolite is the mono, di ortriphosphate of the nucleoside by 5′deuteration of the nucleoside,because 5′-deuteration increases the pool of the nucleosidemonophosphate. Nucleoside monophosphate is metabolized to thediphosphate and/or triphosphate with the corresponding nucleosidediphosphate kinase and then nucleoside triphosphate kinase. This methodis especially useful for nucleosides which are not easilymonophosphorylated, and thus lose substantial activity in the presenceof nucleotidases, that cannot be easily recovered by the action ofnucleoside monophosphate kinase, such as a branched or highlyderivatized nucleoside.

The conversion of nucleoside derivative (A) to nucleoside monophosphate(B) which can then be converted either to inactive nucleoside (C) oractive triphosphate (D) follows.

The efficacy of deuteration can be found indirectly by determining thelevel of nucleoside monophosphate and dephosphorylated nucleosideprovided by a deuterated compound of Formula (I), (II), or (III) ascompared to the level of nucleoside monophosphate and dephosphorylatednucleoside provided by an undeuterated nucleoside monophosphate. Alarger ratio of nucleoside monophosphate to dephosphorylated nucleosideis favorable as a higher percentage of nucleoside monophosphate remainsand can be converted to the active nucleoside triphosphate. Levels offree nucleoside and nucleoside monophosphate in human hepatocytes can bedetermined by an LC/MS assay such as the human hepatocyte LCMS assay ofExample 16.

The efficacy of deuteration can be determined directly by determiningthe level of tri-phosphorylated nucleoside as compared to the level oftriphosphorylated nucleoside provided by an undeuterated nucleosideprodrug. The hepatocyte LCMS assay of Example 16 can also be used todetermine nucleoside triphosphate levels.

The disclosure includes compounds and salts thereof of Formula (I),(II), and (III) and in which the variables meet any of the followingconditions. Any combination of variables is within the scope of thedisclosure as long as a stable compound results.

The Variable Y

Y is NH.

Y is O.

The Variables R₁ and R₂

R₁ is hydroxyl, fluoro, or —OCD₃; R₂ is hydrogen or deuterium; or R₂ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, or C₂-C₆alkynyl; each of which isoptionally deuterated and optionally substituted.

R₁ is hydroxyl or fluoro; and R₂ is C₁-C₄alkyl, allenyl, C₂-C₄alkenyl,or C₂-C₄alkynyl; each of which is optionally deuterated.

R₁ is hydroxyl or fluoro; and R₂ is methyl or —CD₃.

R₁ and R₂ are joined to form a 3- to 6-membered cycloalkyl ring or a 3-to 6-membered heterocycloalkyl ring containing one heteroatom selectedfrom N, O, and S, each of which is optionally substituted.

R₁ and R₂ are joined to form a 3- to 6-membered cycloalkyl ring or a 3-to 6-membered heterocycloalkyl ring containing one heteroatom selectedfrom N, O, and S, each of which is optionally substituted with one ormore substituents independently chosen from halogen, hydroxyl,C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R₁ and R₂ are joined to form a cyclopropyl group.

R₁ is hydroxyl and R₂ is methyl.

R₁ is fluoro and R₂ is methyl.

The R₃ Variable

R₃ is hydrogen, deuterium, halogen, or —N₃ and R₃′ is hydroxyl.

R₃ is C₁-C₄alkyl, allenyl, C₂-C₄alkenyl, C₂-C₄alkynyl,(C₃-C₆cycloalkyl)C₀-C₂alkyl, or (phenyl)ethynyl; and R₃′ is hydroxyl.

R₃ and R₃′ are taken together to form a 3- to 6-membered ring optionallycontaining one heteroatom selected from N, O, and S, which ring isoptionally substituted with one or more substituents independentlyselected from halogen, hydroxyl, C₁-C₂alkyl, and C₁-C₂alkoxy.

R₃ and R₃′ are both hydrogen.

R₃ is —N₃ and R₃′ is hydrogen.

The R₄ and R₅ Variables

R₄ is hydrogen, deuterium, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy; or R₄ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or C₁-C₆alkoxy, each ofwhich is optionally deuterated and optionally substituted; and R₅ ishydrogen or deuterium; or R₅ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, or C₁-C₆alkoxy, each of which is optionally deuterated andoptionally substituted.

R₄ is hydrogen, or deuterium or R₄ is C₁-C₄alkyl, allenyl, C₂-C₄alkenyl,C₂-C₄alkynyl, or C₁-C₄alkoxy, each of which is optionally deuterated;and R₅ is hydrogen or deuterium; or R₅ is C₁-C₆alkyl, allenyl,C₂-C₆alkenyl, C₂-C₆alkynyl, or C₁-C₆alkoxy, each of which is optionallydeuterated.

R₄ and R₅ are both deuterium.

R₄ and R₅ are taken together to form a 3- to 6-membered ring optionallycontaining one heteroatom selected from N, O, and S, which ring isoptionally substituted with one or more substituents independentlyselected from halogen, hydroxyl, C₁-C₂alkyl, and C₁-C₂alkoxy.

The R₆ Variable

R₆ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, each of whichoptionally substituted.

R₆ is (aryl)C₀-C₂alkyl, a 5- to 6-membered monocyclic heteroarylcontaining 1 to 3 heteroatoms independently chosen from N, O, and S, or8- to 10-membered bicyclic heteroaryl containing 1 to 4 heteroatomsindependently chosen from N, O, and S; each of which R₆ is optionallysubstituted.

R₆ is phenyl, pyridyl, naphthyl, or indolyl, each of which is optionallysubstituted with one or more substituents independently chosen fromhalogen, hydroxyl, C₁-C₄alkyl, C₁-C₄alkoxy, (mono- anddi-C₁-C₄alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R₆ is phenyl substituted with one or more substituents independentlychosen from halogen, hydroxyl, amino, cyano, —CHO, —COOH, —CONH₂,C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆alkoxy, C₂-C₆alkanoyl, C₁-C₆alkylester,(mono- and di-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

R₆ is unsubstituted phenyl.

R₆ is unsubstituted naphthyl.

The R₇ and R₈ Variables

R₇ is hydrogen, halogen, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy; or R₇ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆alkoxy,(C₃-C₆cycloalkyl)C₀-C₄alkyl, or (aryl)C₀-C₂alkyl; each of which isoptionally substituted; and R₈ is hydrogen, halogen, C₁-C₂haloalkyl, orC₁-C₂haloalkoxy; or R₈ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, or C₁-C₆alkoxy, each of which is optionally substituted.

R₇ and R₈ are independently chosen from hydrogen, halogen, C₁-C₄alkyl,C₁-C₄alkoxy, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R₇ and R₈ are taken together to form a 3- to 6-membered cycloalkyl ringor 3- to 6-membered heterocycloalkyl ring containing one heteroatomchosen from N, O, and S; each of which is optionally substituted.

R₇ is C₁-C₆alkyl and R₈ is hydrogen, halogen, or C₁-C₆alkyl.

R₇ is methyl and R₈ is hydrogen.

The R₉ Variable

R₉ is C₁-C₆alkyl, (C₃-C₇cycloalkyl)C₀-C₄alkyl, or (phenyl)C₀-C₄alkyl,each of which is optionally substituted.

R₉ is C₁-C₆alkyl.

R₉ is (C₃-C₇cycloalkyl)C₀-C₂alkyl or (phenyl)C₀-C₂alkyl, each of whichis optionally substituted with one or more substituents independentlychosen from halogen, hydroxyl, C₁-C₄alkyl, C₁-C₄alkoxy, (mono- anddi-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

The B₁ Variable

B₁ is a pyrimidine base chosen from group

The disclosure includes compounds of Formula (I), (II), or (III) inwhich B₁ is selected from the above group and any of the followingconditions are met.

R₁₂ is hydrogen and R₁₃ is deuterium.

R₁₂ is deuterium and R₁₂ is hydrogen.

R₁₂ and R₁₃ are both hydrogen.

R₁₂ and R₁₃ are both deuterium.

R₁₂ is CD₃ and R₁₃ is deuterium.

R₁₂ is CD₃ and R₁₂ is hydrogen.

R₁₂ is hydrogen, R₁₃ is deuterium, and R₁₄ is amino.

R₁₂ is hydrogen, R₁₃ is deuterium, and R₁₄ is hydroxyl.

R₁₂ and R₁₃ are both deuterium and R₁₄ is hydroxyl.

R₁₂ and R₁₃ are both hydrogen and R₁₄ is hydroxyl.

R₁₂ and R₁₃ are both hydrogen and R₁₄ is amino.

R₁₂ and R₁₃ are both deuterium and R₁₄ is amino.

R₁₂ is hydrogen, R₁₃ is deuterium, and R₁₄ is hydroxyl.

OTHER EMBODIMENTS

The disclosure includes compounds of Formula (I), (II), or (III) inwhich the following conditions are met.

R₁ is hydroxyl, fluoro, or —OCD₃; and R₂ is hydrogen, —CH₃, or —CD₃.

Or, R₁ and R₂ are joined to form a cyclopropyl.

R₃ is hydrogen or —N₃.

R₄ and R₅ are independently hydrogen, deuterium, methyl, or —CD₃.

R₆ is phenyl, pyridyl, naphthyl, or indolyl, each of which is optionallysubstituted with one or more substituents independently chosen fromhalogen, hydroxyl, amino, cyano, —CHO, —COOH, —CONH₂, C₁-C₆alkyl,C₂-C₆alkenyl, C₁-C₆alkoxy, C₂-C₆alkanoyl, C₁-C₆alkylester, (mono- anddi-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R₇ is hydrogen, halogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₂haloalkyl,C₁-C₂haloalkoxy, (C₃-C₆cycloalkyl)C₀-C₄alkyl, or (aryl)C₀-C₂alkyl; andR₈ is hydrogen, halogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₂haloalkyl, orC₁-C₂haloalkoxy.

Or, R₇ and R₈ are taken together to form a 3- to 6-membered cycloalkylring.

R₉ is C₁-C₆alkyl, (C₃-C₇cycloalkyl)C₀-C₄alkyl, or (aryl)C₀-C₄alkyl, eachof which is optionally substituted with one or more substituentsindependently chosen from halogen, hydroxyl, amino, cyano, —CHO, —COOH,—CONH₂, C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆alkoxy, C₂-C₆alkanoyl,C₁-C₆alkylester, (mono- and di-C₁-C₆alkylamino)C₀-C₂alkyl,C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

B₁ is a base selected from

For B₁ the following conditions are met:

R₁₂ and R₁₃ are independently hydrogen and deuterium.

R₁₂ and R₁₃ are independently hydrogen, deuterium, and methyl.

R₁₄ is hydroxyl, amino; C₁-C₄alkoxy, C₁-C₄alkylester, or mono- ordi-C₁-C₄alkylcarbamate; and in the compound of Formula (I), (II), or(III) each position represented by D has a deuterium enrichment of atleast 50%. One or more of R₁, R₂, R₄, R₅, R₁₀, R₁₁, R₁₂, R₁₃, and R₁₄ isdeuterium with a deuterium enrichment of at least 50% % or a deuteratedsubstituent with at least one position having a deuterium enrichment ofat least 50%.

The disclosure also includes compounds and salts of Formula (I), (II),or (III), in which: B₁ is uridine or cytosine:

In certain embodiments in which B₁ is uridine, R₁₂ and R₁₃ are bothdeuterium; R₁₂ is deuterium and R₁₃ is hydrogen; R₁₂ is hydrogen and R₁₃is deuterium; or R₁₂ and R₁₃ are both hydrogen. In certain embodiment inwhich B₁ is cytosine R₁₂ is CD₃ and R₁₃ is deuterium.

R₁₂ and R₁₃ are both deuterium and R₁₄ is hydroxyl.

R₁ is hydroxyl, fluoro, or —OCD₃; and R₂ is hydrogen, —CH₃, or —CD₃.

Or, R₁ and R₂ are joined to form a cyclopropyl.

R₃ is hydrogen or —N₃.

R₄ and R₅ are independently hydrogen, deuterium, methyl, or —CD₃.

R₆ is phenyl, pyridyl, naphthyl, or indolyl, each of which is optionallysubstituted with one or more substituents independently chosen fromhalogen, hydroxyl, C₁-C₄alkyl, C₁-C₄alkoxy, (mono- anddi-C₁-C₄alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R₇ is hydrogen, halogen, C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₂haloalkyl,C₁-C₂haloalkoxy, (C₃-C₆cycloalkyl)C₀-C₂alkyl, or (phenyl)C₀-C₂alkyl; R₈is hydrogen, halogen, C₁-C₂alkyl, or C₁-C₂alkoxy.

R₇ and R₈ are taken together to form a 3- to 6-membered cycloalkyl ring;and

R₉ is C₁-C₆alkyl, (C₃-C₇cycloalkyl)C₀-C₂alkyl, or (aryl)C₀-C₂alkyl, eachof which is optionally substituted with one or more substituentsindependently chosen from halogen, hydroxyl, C₁-C₄alkyl, C₁-C₄alkoxy,(mono- and di-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

The disclosure includes compounds and salts of Formula (I), (II), and(III) in which:

R₆ is phenyl, naphthyl, or indolyl;

R₇ is hydrogen, halogen, or C₁-C₄alkyl;

R₈ is hydrogen, halogen, C₁-C₂alkyl, or C₁-C₂alkoxy; and

R₉ is C₁-C₆alkyl.

This disclosures include compounds, and the pharmaceutically acceptablesalts thereof of Formula (IV)

In certain compounds and salts of Formula (IV) B₁ is

In certain compounds and salts of Formula (IV) the following conditionsare met.

R₄ and R₅ are both deuterium; one of R₁₂ and R₁₃ is deuterium and theother is hydrogen, or R₁₂ is CD₃ and R₁₃ is hydrogen or deuterium.

R₁ is hydroxyl and R₂ is methyl.

R₁ is hydroxyl and R₂ is —CD₃.

R₁ and R₂ are joined to form a cyclopropyl group.

R₁ is fluoro and R₂ is methyl or —CD₃.

The disclosure includes the following compounds

A compound or salt thereof of claim 1, wherein the compound is chosenfrom:

Pharmaceutical Compositions

This disclosure also includes pharmaceutical compositions andcombinations comprising a compound of Formula (I) and at least oneadditional active agent, as well as methods of treatment comprisingadministering such compositions to a patient infected with hepatitis C.In certain embodiments the additional active agent is an HCV HS3protease inhibitor or an HCV NS5a inhibitor.

For example, in some embodiments, the additional active agent issovaprevir or ACH-2684 (HCV NS3 protease inhibitors) and/or an (NS5ainhibitor).

The disclosure includes compositions in which the additional activeagent is a NS3 inhibitor such as Sovaprevir or ACH-2684

The disclosure also includes compositions which contain an additionalactive agent, such as a NS5A inhibitor chosen from:

NS3 protease inhibitors, useful in the pharmaceutical compositions andcombinations described here have been disclosed previously, for examplein U.S. Pat. No. 7,906,619, issued Mar. 15, 2011, is hereby incorporatedby reference in its entirety for its teachings regarding4-amino-4-oxobutanoyl peptides. The '619 patent is particularlyincorporated by reference at the Examples section beginning in column 50and extending to column 85 which discloses compounds useful incompositions/combination with Compounds of Formula (I) described here.

US Pat. Appl. No. 2010-0216725, published Aug. 26, 2010, is herebyincorporated by reference in its entirety for its teachings regarding4-amino-4-oxobutanoyl peptides. The '725 application is particularlyincorporated by reference at the Examples section beginning at page 22and extending to page 100 which discloses compounds useful incompositions/combination with Compounds of Formula (I) described here.

US Pat. Appl. No. 2010-0152103, published Jun. 17, 2010, herebyincorporated by reference in its entirety for its teachings regarding4-amino-4-oxobutanoyl peptide cyclic analogues. The '103 application isparticularly incorporated by reference at the Examples section beginningat page 19 and extending to page 60 which discloses compounds useful incompositions/combination with Compounds of Formula (I) described here.Particularly the compounds of Formula (I) disclosed herein may be usedin combination with an NS3 protease inhibitor.

NS5a inhibitors, useful in the pharmaceutical compositions andcombinations described here have been disclosed previously. U.S. Pat.Pub. No. US-2012-0302528, published Nov. 29, 2012, is herebyincorporated by reference in its entirety for its teachings regardingNs5a Inhibitors. Particular NS5a inhibitors that may be used combinationwith the compound of this disclosure include compounds of the formulaT-R-J¹-W-A-W-J¹-R-T; T-R-J¹-A-J¹-R-T; T-R-J²-A-J²-R-T; orT-R-J¹-W-A-J¹-R-T.

In the above formulae, T is independently chosen at each occurrence andis T^(k) where k is an integer from 1 to 2.

T¹ is —Y—Z, where Y is covalently bound to R and Y is a bond orC₁-C₄alkylene optionally substituted with oxo; and Z is a 5 or6-membered heterocyclic group, each of which T¹ is substituted with (i)at least one substituent selected from —(C═O)OH, —(C═O)NH₂, —(C═O)H,—C₁-C₄alkoxy, C₂-C₄alkanoyl, C₁-C₄alkylester, C₁-C₄alkenylester, andmono- and di-C₁-C₄alkylcarboxamide and (ii) optionally substituted withone or more substituents independently chosen from halogen, hydroxyl,C₁-C₂alkyl, and C₁-C₂alkoxy.

T² is independently chosen at each occurrence from C₂-C₆alkanoyl,C₁-C₆alkylester, C₁-C₆alkenylester, C₁-C₆alkylsulfonamide,C₁-C₆alkylsulfonyl, C₂-C₆alkanoyl substituted with mono- ordi-C₁-C₆hydrocarbylcarbamate, C₂-C₆alkanoyl substituted with urea ormono- or di-C₁-C₆alkylurea, and C₂-C₆alkanoyl substituted with mono- ordi-C₁-C₆alkylcarboxamide, each of which T² is optionally substitutedwith 1 or more substituents independently chosen from amino, cyano,hydroxyl, halogen, (C₁-C₄alkoxy)C₀-C₄alkyl, (mono- anddi-C₁-C₄alkylamino)C₀-C₄alkyl, C₁-C₆alkyl, (C₁-C₄thioalkyl)C₀-C₄alkyl,C₃-C₇cycloalkyl, phenyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R is independently chosen at each occurrence from 4- to 6-membered ringscontaining one or two nitrogen atoms with remaining ring atoms beingcarbon, which R is saturated or contains 1 unsaturated bond and isoptionally bridged with an methylene or ethylene bridge, or fused to aphenyl or 5- to 6-membered heteroaryl ring; and 6- to 10-membered fusedor spiro bicyclic ring systems containing one or two nitrogen atoms withremaining ring atoms being carbon, which 6- to 10-membered bicyclic ringis saturated or contains 1 unsaturated bond.

Each R is optionally substituted with one or more substituentsindependently chosen from cyano, hydroxyl, halogen, C₁-C₂alkyl,C₁-C₂alkoxy, C₁-C₂haloalkyl, C₁-C₂haloalkyl, C₁-C₂haloalkylene, andC₁-C₂alkylsulfonyl;

J¹ is phenyl or a 5- to 6-membered heteroaryl group, such as a group,where each J¹ is optionally substituted with one or more substituentsindependently chosen from amino, cyano, hydroxyl, halogen, C₁-C₄alkyl,C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

J² is a 8- to 10-membered bicyclic heteroaryl group containing 1 to 4heteroatoms independently chosen from N, O, and S, such as abenzimidazole group, wherein J² is optionally substituted with one ormore substituents independently chosen from amino, cyano, hydroxyl,halogen, C₁-C₄alkyl, C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino,C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

W is independently chosen at each occurrence and is a phenyl, pyridyl oralkynyl group, optionally substituted with one or more substituentsindependently chosen from amino, cyano, hydroxyl, halogen, C₁-C₄alkyl,C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

A is a [2.2]-cyclophane, where each 2 atom linker of the[2.2]-cyclophane optionally contains a heteroatom selected from N, O, orS and is optionally substituted with 1 oxo group, and one or moresubstituents independently chosen from halogen, hydroxy, amino,C₁-C₂alkyl, and C₁-C₂alkoxy; or

A is a group of the formula

wherein Q is a neutral or cationic metal, each of which A is optionallysubstituted with one or more substituents independently chosen fromhalogen, C₁-C₂alkyl, and C₁-C₂alkoxy; or

A is a group of the formula

which A is optionally substituted with one or more substituentsindependently chosen from halogen, C₁-C₂alkyl, and C₁-C₂alkoxy.

The disclosure particularly includes pharmaceutical compositionscontaining one deuterated nucleoside prodrug of Formula (I), one NS3protease inhibitor, and one NS5a inhibitor.

In certain embodiments the deuterated nucleoside prodrug is

The NS3 protease inhibitor is chosen from

The NS5a inhibitor is chosen from

Synthetic Methods and Intermediates

The disclosure includes methods of preparing compounds of Formula (I).

comprising(i) reacting an amino ester (1) with a dichlorophosphate (2) to form areaction mixture;

(ii) adding to the reaction mixture of (i) an aryl hydroxyl, arylsulfhydryl, or hydroxylimide, Ar-LH, where L is S or O, and Ar-LH is

to form an intermediate (3)

and(iii) reacting the intermediate (3) with a nucleoside

to form the compound of Formula (I). The variables B₁, R₁, R₂, R₃, R₃′,R₄, R₅, R₆, R₇, R₈, and R₉ may carry the definitions set forth in theSUMMARY section or for any of the embodiments of Formula (I) describedherein.

In one embodiment the disclosure provides a method for making a compoundof Formula (I), in which the compound of Formula (I) is

comprising(i) reacting an amino ester (1), wherein the amino ester is L-alanineisopropyl ester, with a dichlorophosphate (2), wherein thedichlorophosphate is phenoxydichlorophosphate, to form a reactionmixture;

(ii) adding to the reaction mixture of (i) an aryl hydroxyl, arylsulfhydryl, or hydroxylimide, Ar-LH, where L is S or O, and aryl is anoptionally substituted aryl, heteroaryl, or heterocycloalkyl group suchas phenyl, pyrrole, pyridyl, or indole, and in certain embodiments Ar-LHis

to form an intermediate (3)

and(iii) reacting the intermediate (3) with a nucleoside (14)

to form

In certain embodiments the amino ester (1) and the dichlorophosphate (2)are combined at a temperature less than −20° C., more preferably at atemperature of about −40° C. to about −60° C.

In certain embodiments triethylamine or other base is added to themixture of amino ester (1) and the dichlorophosphate (2). In certainembodiments the addition occurs in an organic solvent, such asdichloromethane, or other organic solvent such as 1-propanol,2-methyltetrahydrofuran, or tetrahydrofuran.

Aryl hydroxyl or aryl sulfhydryl is added to the reaction mixture formedby the combination of amino ester (1) and dichlorophosphate (2). Incertain embodiments the aryl hydroxyl or aryl sulfhydryl istrichlorothiophenol, but may also be replaced by other groups such asnitrothiophenol, bromothiophenol, N-hydroxysuccinamide,N-hydroxyphthalimide, or nitrohydroxypyridine. In certain embodimentsthe aryl hydroxyl or aryl sulfhydryl is added as a solution indichloromethane or other organic solvent such as 1-propanol,2-methyltetrahydrofuran, or tetrahydrofuran. In certain embodiments thesolution containing the aryl hydroxyl or aryl sulfhydryl also containstriethylamine or other base. After the aryl hydroxyl or aryl sulfhydrylis added to the reaction mixture formed by the combination of aminoester (1) and dichlorophosphate (2) the resulting solution can be warmedto a temperature above 0° C., above 15° C., and preferably to about 20°C. to about 35° C. and may be stirred at this temperature for a periodof from about 5 hours to about 30 hours and more preferably from about10 hours to about 20 hours or about 15 hours.

The reaction mixture formed by the addition of aryl hydroxyl or arylsulfhydryl to amino ester (1) and dichlorophosphate (2) may be extractedwith water, which is optionally saturated with salt such as sodiumbicarbonate or ammonium sulfate. The crude intermediate (3) obtained bydrying the organic fraction may be purified by column chromatography,recrystallization, or other suitable purification method. The desiredisomer of the intermediate (3) may be obtained by dissolving theintermediate, preferably after purification, in ethyl acetate/heptane orother mixture of other non-polar/polar aprotic solvent such as a mixtureof heptane, cyclohexane, benzene (non-polar solvents) and THF, DMF, orDCM (polar aprotic solvents) and seeding the solution with a smallamount of the desired isomer of intermediate (3). (This seed amount of(3) may have been obtained by another method.)

The nucleoside (14) may be suspended in a solvent, preferably a nonpolaraprotic solvent such as THF, DCM, or DMF. The suspension of nucleoside(14) in solvent may cooled below 0° C., preferably below −10° C. toabout −40° C., and preferably to about −20° C.

The suspension of nucleoside (14) in solvent may be added to analkylating agent such as a Grignard reagent, for example tert-butylMgCl, or other alkylmetal halide, at a temperature below 0° C.,preferably below −10° C. to about −40° C., and preferably to about −20°C. The reaction mixture of nucleoside (14) in solvent and alkylatingagent is warmed to above 0° C., and preferably to about 20° C. to about30° C., and stirred for about 1 to about 5 hours, or for preferably fromabout 2 to about 3 hours. The reaction mixture may then be cooled againto below 0° C., preferably below −5° C. to about −20° C., and preferablyto about −10° C. Intermediate (3), which may optionally be opticallypure, is added to the reaction mixture containing the nucleoside (14).The reaction mixture of intermediate (3) and nucleoside (14) is warmedto above 0° C., and preferably to about 20° C. to about 30° C., andstirred for at least 5 hours, preferably about 10 to about 20 hours, orpreferably about 15 hours. The reaction may be cooled to about 0° C. andquenched with acid, such as HCl or other acid capable of providing a pHof approximately 1 to 3 or preferably about 2. The resulting product, acompound of Formula (I), may then be purified by organic phaseextraction, column chromatography, HPLC, crystallization or any othersuitable purification method.

In addition to a method of making a compound of Formula (I) thedisclosure provides an intermediate useful for making a compound ofFormula (I) of the formula

where the variables Ar, L, R₆, R₇, R₈, and R₉ carry the definitions setfor these variables earlier in this section, or R₆, R₇, R₈, and R₉ maycarry any of the values set for forth in this disclosure for R₆, R₇, R₈,and R₉. In certain embodiments the intermediate is

In other embodiments the intermediate is

Compounds of the disclosure can also be made by the method shown inreaction Scheme I.

In reaction Scheme I a pyrimidine base ii, which has up to threesubstituents and may or may not contain deuterium, is reacted withtetrabenzoyl sugar i to give nucleoside iii. Nucleoside iii is thentreated with 2,2-dimethoxypropane and p-toluene sulfonic acid (p-TSA) togive acetonide iv. Acetonide iv is treated with pyridinium dichromate(PDC) in t-butanol to effect oxidation and esterification to t-butylester v. Compound v is reacted with sodium borodeuteride indeuteroethanol to provide the dideuterated compound vi, which is thentreated with hydrochloric acid to remove the acetonide and provide triolvii. Compound vii is treated with a base such as t-butyl magnesiumhalide, followed by addition of an activated phosphate derivative viiito afford deuterated nucleoside phosphoramidate ix as a final product.

Pharmaceutical Preparations

Compounds disclosed herein can be administered as the neat chemical, butare preferably administered as a pharmaceutical composition.Accordingly, the disclosure provides pharmaceutical compositionscomprising a compound or pharmaceutically acceptable salt of Formula(I), together with at least one pharmaceutically acceptable carrier. Thepharmaceutical composition/combination may contain a compound or salt ofFormula (I) as the only active agent, but is preferably contains atleast one additional active agent. In certain embodiments it ispreferred that the additional active agent is an NS3 protease inhibitoror NS5a inhibitor. In certain embodiments the pharmaceutical compositionis in a dosage form that contains from about 0.1 mg to about 2000 mg,from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, orfrom about 200 mg to about 600 mg of a compound of Formula (I) andoptionally from about 0.1 mg to about 2000 mg, from about 10 mg to about1000 mg, from about 100 mg to about 800 mg, or from about 200 mg toabout 600 mg of an additional active agent in a unit dosage form. Thepharmaceutical composition may also include a molar ratio of a compoundof Formula (I) and an additional active agent. For example thepharmaceutical composition may contain a molar ratio of about 0.5:1,about 1:1, about 2:1, about 3:1 or from about 1.5:1 to about 4:1 of anNS3 protease inhibitor.

Compounds disclosed herein may be administered orally, topically,parenterally, by inhalation or spray, sublingually, transdermally, viabuccal administration, rectally, as an ophthalmic solution, or by othermeans, in dosage unit formulations containing conventionalpharmaceutically acceptable carriers. The pharmaceutical composition maybe formulated as any pharmaceutically useful form, e.g., as an aerosol,a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermalpatch, or an ophthalmic solution. Some dosage forms, such as tablets andcapsules, are subdivided into suitably sized unit doses containingappropriate quantities of the active components, e.g., an effectiveamount to achieve the desired purpose.

Carriers include excipients and diluents and must be of sufficientlyhigh purity and sufficiently low toxicity to render them suitable foradministration to the patient being treated. The carrier can be inert orit can possess pharmaceutical benefits of its own. The amount of carrieremployed in conjunction with the compound is sufficient to provide apractical quantity of material for administration per unit dose of thecompound.

Classes of carriers include, but are not limited to binders, bufferingagents, coloring agents, diluents, disintegrants, emulsifiers,flavorants, glidents, lubricants, preservatives, stabilizers,surfactants, tableting agents, and wetting agents. Some carriers may belisted in more than one class, for example vegetable oil may be used asa lubricant in some formulations and a diluent in others. Exemplarypharmaceutically acceptable carriers include sugars, starches,celluloses, powdered tragacanth, malt, gelatin; talc, and vegetableoils. Optional active agents may be included in a pharmaceuticalcomposition, which do not substantially interfere with the activity ofthe compound of the present invention.

The pharmaceutical compositions/combinations can be formulated for oraladministration. These compositions contain between 0.1 and 99 weight %(wt. %) of a compound of Formula (I) and usually at least about 5 wt. %of a compound of Formula. Some embodiments contain from about 25 wt. %to about 50 wt. % or from about 5 wt. % to about 75 wt. % of thecompound of Formula.

Methods of Treatment

The pharmaceutical compositions/combinations disclosed herein are usefulfor treating viral infections in patients. In one embodiment the viralinfection is a hepatitis C infection but the infection may also be anRNA viral infection, a such as a Togaviridae, Picornaviridae,Coronaviridae, or Flaviviridae viral infection. The disclosure includesa method of treating a Togaviridae, Picornaviridae, Coronaviridae, orFlaviviridae viral infection by administering a compound of Formula (I),to a subject infected with a togavirus, picornavirus, coronavirus, orflavivirus. Flaviviridae viral infections include infections withviruses of the genera Flavivirus, Pestivirus, and Hepacivirus.Flavivirus infections include yellow fever, Dengue fever, West Nilevirus, encephalitis, including St. Louis encephalitis, Japanese Bencephalitis, California encephalitis, central European encephalitis,Russian spring-summer encephalitis, and Murray Valley encephalitis,Wesselsbron disease, and Powassan disease. Pestivirus infections includeprimarily livestock diseases, including swine fever in pigs, BVDV(bovine viral diarrhea virus) in cattle, and Border Disease virusinfections. Hepacivirus infections includes Hepatitis C and canineHepacivirus. Togavirus infections include Sindbis virus, Eastern equineencephalitis virus, Western equine encephalitis virus, Venezuelan equineencephalitis virus, Ross River virus, O'nyong'nyong virus, Chikungunyavirus, Semliki Forest virus, and Rubella virus. Picornavirus infectionsinclude infections with viruses of the genuses Aphthovirus, Aquamavirus,Avihepatovirus, Cardiovirus, Cosavirus, Dicipivirus, Enterovirus,Erbovirus, Hepatovirus, Kobuvirus, Megrivirus, Parechovirus, Salivirus,Sapelovirus, Senecavirus, Teschovirus, and Tremovirus. Coronavirusinfections include infections with virus of the genusesAlphacoronavirus, Betacoronavirus (which includes Severe acuterespiratory coronavirus (SARS)), Gammacoronavirus, and Deltacoronavirus.The disclosure particularly includes compositions comprising a compoundof Formula (I) useful for treating Dengue fever, West Nile fever, yellowfever, or BVDV (bovine viral diarrhea virus) and methods of treatingthese infections by administering a compound of Formula (I) to a patientinfected with the virus.

This disclosure provides methods of treating viral infections, includinghepatitis C infections, by providing an effective amount of a compoundor pharmaceutically acceptable salt of Formula (I) to patient infectedwith a hepatitis C virus. A compound or salt of Formula (I) may beprovided as the only active agent or may be provided together with oneor more additional active agents. In certain embodiments the compound orsalt of Formula (I) is administered together with a NS3 proteaseinhibitor and/or NS5a inhibitor.

An effective amount of a pharmaceutical composition/combination of theinvention may be an amount sufficient to (a) inhibit the progression ofhepatitis C; (b) cause a regression of the hepatitis C infection; or (c)cause a cure of a hepatitis C infection such that HCV virus or HCVantibodies can no longer be detected in a previously infected patient'sblood or plasma. An amount of a pharmaceutical composition/combinationeffective to inhibit the progress or cause a regression of hepatitis Cincludes an amount effective to stop the worsening of symptoms ofhepatitis C or reduce the symptoms experienced by a patient infectedwith the hepatitis C virus. Alternatively a halt in progression orregression of hepatitis C may be indicated by any of several markers forthe disease. For example, a lack of increase or reduction in thehepatitis C viral load or a lack of increase or reduction in the numberof circulating HCV antibodies in a patient's blood are markers of a haltin progression or regression of hepatitis C infection. Other hepatitis Cdisease markers include aminotransferase levels, particularly levels ofthe liver enzymes AST and ALT. These levels will typically be elevatedin a HCV infected patient. Disease regression is usually marked by thereturn of AST and ALT levels to the normal range.

Symptoms of hepatitis C that may be affected by an effective amount of apharmaceutical composition/combination of the invention includedecreased liver function, fatigue, flu-like symptoms: fever, chills,muscle aches, joint pain, and headaches, nausea, aversion to certainfoods, unexplained weight loss, psychological disorders includingdepression, tenderness in the abdomen, and jaundice.

“Liver function” refers to a normal function of the liver, including,but not limited to, a synthetic function including synthesis of proteinssuch as serum proteins (e.g., albumin, clotting factors, alkalinephosphatase, aminotransferases (e.g., alanine transaminase, aspartatetransaminase), 5′-nucleosidase, glutaminyl transpeptidase, etc.),synthesis of bilirubin, synthesis of cholesterol, and synthesis of bileacids; a liver metabolic function, including carbohydrate metabolism,amino acid and ammonia metabolism, hormone metabolism, and lipidmetabolism; detoxification of exogenous drugs; and a hemodynamicfunction, including splanchnic and portal hemodynamics.

An effective amount of a pharmaceutical composition/combinationdescribed herein will also provide a sufficient concentration of theactive agents in the concentration when administered to a patient. Asufficient concentration of an active agent is a concentration of theagent in the patient's body necessary to prevent or combat theinfection. Such an amount may be ascertained experimentally, for exampleby assaying blood concentration of the agent, or theoretically, bycalculating bioavailability. The amount of an active agent sufficient toinhibit viral infection in vitro may be determined with a conventionalassay for viral infectivity such as a replicon based assay, which hasbeen described in the literature.

Pharmaceutical compositions/combinations and methods of treatment inwhich a compound or salt of Formula (I) is provided together with one ormore additional active agents are included herein. In preferredembodiments a compound of Formula (I) is provided together with an NS3protease inhibitor, and/or NS5a protease inhibitor, either in a singlepharmaceutical composition or a in separate dosage forms withinstructions to the patient to use the compound of Formula (I) andadditional active agent together. Compounds disclosed in U.S. Pat. No.7,906,619, US Pat. Pub. No. 2010-0216725, and US Pat. Pub. No.2010-0152103 are suitable NS3 protease inhibitors for use in combinationwith compounds and salts of Formula (I). Compounds disclosed in US Pat.Appl. No. 2012-0302538 are suitable NS5a inhibitors for use incombination with compounds of Formula (I). In certain embodiments theactive agent (or agents) is an HCV protease inhibitor or HCV polymeraseinhibitor. For example the protease inhibitor may be telaprevir (VX-950)and the polymerase inhibitor may be valopicitabine, or NM 107, theactive agent which valopicitabine is converted into in vivo. In certainembodiments the at least one additional active agent is ribavirin,interferon, or Peg-interferon alpha conjugate. In certain embodimentsthe at least one additional active agent is sovaprevir, ACH-2684, orACH-3102.

The compound or pharmaceutically acceptable salt of Formula (I) and atleast one additional active agent may be: (1) co-formulated andadministered or delivered simultaneously in a combined formulation; (2)delivered by alternation or in parallel as separate formulations; or (3)by any other combination therapy regimen known in the art. Whendelivered in alternation therapy, the methods of the invention maycomprise administering or delivering the compound or salt of Formula (I)and an additional active agent sequentially, e.g., in separate solution,emulsion, suspension, tablets, pills or capsules, or by differentinjections in separate syringes. In general, during alternation therapy,an effective dosage of each active ingredient is administeredsequentially, i.e., serially, whereas in simultaneous therapy, effectivedosages of two or more active ingredients are administered together.Various sequences of intermittent combination therapy may also be used.

Methods of treatment and pharmaceutical combinations including compoundsor pharmaceutically acceptable salts of Formula (I) described hereintogether with any one or combination of the following compounds andsubstances as an additional active agent are provided by the disclosure:

Caspase Inhibitors: IDN-6556 (Idun Pharmaceuticals) and GS-9450 (Gilead)

Cyclophilin Inhibitors: for example, NIM811 (Novartis), SCY-635(Scynexis), and DEBIO-025 (Debiopharm);

Cytochrome P450 monooxygenase inhibitors: ritonavir, ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole,cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine,fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir,fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, andVX-497 (Merimebodib). Preferred CYP inhibitors include ritonavir,ketoconazole, troleandomycin, 4-methyl pyrazole, cyclosporin, andclomethiazole;

Entry Inhibitors: ITX-5061 (iTherX)

Glucocorticoids: hydrocortisone, cortisone, prednisone, prednisolone,methylprednisolone, triamcinolone, paramethasone, betamethasone, anddexamethasone.

HCV Protease Inhibitors: for example Sovaprevir and ACH-2684. ABT-450(Abbott), ACL-181 and AVL-192 (Avila), BI-335 (Boehringer Ingelheim),BMS-032 (Bristol Myers Squibb), Boceprevir (Merck), danoprevir(Hoffman-La Roche and Genentech), TMC435 (Merck), GS-9256 (Gilead),GS-9451 (Gilead), R7227 (Intermune), Telaprevir (VX-950, Vertex), VX-985(Vertex), Simeprevir (TMC435, Tibotec), Fosamprenavir (prodrug ofAmprenavir, Glaxo/Vertex), indinavir (CRIXIVAN, Merck), TMC435350(Tibotec/Medivir), Faldaprevir (BI 201335. Boehringer Ingelheim),PHX-1766 (Phenomix), Vaniprevir (, MK-7009, Merck), narlaprevir(SCH900518, Schering), MK-5172 (Merck)

Hematopoietins: hematopoietin-1 and hematopoietin-2. Other members ofthe hematopoietin superfamily such as the various colony stimulatingfactors (e.g., G-CSF, GM-CSF, M-CSF), Epo, and SCF (stem cell factor)

Homeopathic Therapies: Milk Thistle, silymarin, ginseng, glycyrrhizin,licorice root, schisandra, vitamin C, vitamin E, beta carotene, andselenium

Immunomodulatory compounds: thalidomide, IL-2, hematopoietins, IMPDHinhibitors, for example Merimepodib (Vertex Pharmaceuticals Inc.),interferon, including natural interferon (such as OMNIFERON, Viragen andSUMIFERON, Sumitomo, a blend of natural interferons), natural interferonalpha (ALFERON, Hemispherx Biopharma, Inc.), interferon alpha-n1 fromlymphblastoid cells (WELLFERON, Glaxo Wellcome), oral alpha interferon,Peg-interferon, Peg-interferon alfa 2a (PEGASYS, Roche), recombinantinterferon alfa 2a (ROFERON, Roche), inhaled interferon alpha 2b (AERX,Aradigm), Peg-interferon alpha 2b (ALBUFERON, Human GenomeSciences/Novartis, PEGINTRON, Schering), recombinant interferon alfa 2b(INTRON A, Schering), pegylated interferon alfa 2b (PEG-INTRON,Schering, VIRAFERONPEG, Schering), interferon beta-1a (REBIF,Ares-Serono, Inc. and Pfizer), consensus interferon alpha (INFERGEN,Intermune), interferon gamma-1b (ACTIMMUNE, Intermune, Inc.),un-pegylated interferon alpha, alpha interferon, and its analogs, andsynthetic thymosin alpha 1 (ZADAXIN, SciClone Pharmaceuticals Inc.), andlamdba interferon (BMS)

Immunosupressants: sirolimus (RAPAMUNE, Wyeth)

Interleukins: (IL-1, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12), LIF,TGF-beta, TNF-alpha) and other low molecular weight factors (e.g.AcSDKP, pEEDCK, thymic hormones, and minicytokines)

Interferon Enhancers: EMZ702 (Transition Therapeutics)

IRES inhibitors: VGX-410C (VGX Pharma)

Monoclonal and Polyclonal antibodies: XTL-6865 (HEPX-C, XTL), HuMax-HepC(Genmab), Hepatitis C Immune Globin (human) (CIVACIR, NabiBiopharmceuticals), XTL-002 (XTL), Rituximab (RITUXAN, Genentech/IDEC),GS-6624 (Gilead)

Nucleoside analogues: IDX-184 (Idenix), Sofosbuvier (PSI-7977,Pharmasset and Gilead), PSI-938 (Pharmasset), R7128 (Roche), R7348(Roche), GS-6620 (Gilead), TMC-649 (Tibotec), Lamivudine (EPIVIR, 3TC,GlaxoSmithKline), MK-0608 (Merck), zalcitabine (HIVID, Roche USPharmaceuticals), ribavirin (including COPEGUS (Roche), REBETOL(Schering), VILONA (ICN Pharmaceuticals, and VIRAZOLE (ICNPharmaceuticals), isatoribine (Anadys Pharmaceuticals), ANA971 (AnadysPharmaceuticals), ANA245 (Anadys Pharmaceuticals), and viramidine (ICN),an amidine prodrug of ribavirin. Combinations of nucleoside analoguesmay also be employed.

Non-nucleoside inhibitors: PSI-6130 (Roche/Pharmasset), ABT-333 andABT-072 (Abbott), delaviridine (RESCRIPTOR, Pfizer), PF-868554 (Pfizer),GSK-852 (GlaxoSmithKline), IDX-325 (Idenix), Setrobuvir (ANA-598,Anadys), VX-222 (Vertex), MK-3281 (Merck), BI-127 (BoehringerIngelheim), BMS-325 (Bristol Myers), and HCV-796 (Viropharm)

NS4b inhibitors: clemizole (Eiger BioPharmaceuticals, Inc.)

NS5a inhibitors: A-382 (Arrow Therapeutics), Daclatasvir (BMS-790052,BMS), AZD-7295 (Astra Zeneca); PPI-461 (Presidio), PPI-688 (Presidio),IDX719 (Idenix), IDX184 (Idenix)

NS5b inhibitors: INX-181, MBX-700 (Microbotix/Merck), MK-3281, PSI-7977,PSI-7851, PSI-938, RG-9190, VX-222 (Vertex), and BMS-791325 (BristolMyers Squibb).

P7 protein inhibitor: amantadine (SYMMETREL, Endo Pharmaceuticals, Inc.)

Polymerase inhibitors: NM283 (valopicitabine) (Idenix), JTK 003 (AKROSPharma), HCV-796 (ViroPharma/Wyeth), RG7128 (Mericitabine, Genentech),R1626 (Roche), PSI-7851 (Pharmasset), ANA598 (Anadys), BI207127(Boehringer-Ingelheim), Tegobuvir (GS 9190, Gilead), VX-135 (Vertex,Alios).

RNA interference: SIRNA-034 RNAi (Sirna Therapeutics) and ISI 14803(Isis Pharmaceutical/Elan)

Therapeutic Vaccines: IC41 (Intercell), IMN-0101 (Imnogenetics), GI 5005(Globeimmune), Chronvac-C (Tripep/Inovio), ED-002 (Imnogenetics),Hepavaxx C (ViRex Medical)

TNF agonists: adalimumab (HUMIRA, Abbott), entanercept (ENBREL, Amgenand Wyeth), infliximab (REMICADE, Centocor, Inc.)

Tubulin inhibitors: Colchicine

Sphingosine-1-phosphate receptor modulators: FTY720 (Novartis)

TLR agonists: ANA-975 (Anadys Pharmaceuticals), TLR7 agonist (AnadysPharmaceuticals), CPG10101(Coley), and TLR9 agonists including CPG 7909(Coley).

Vaccines: HCV/MF59 (Chiron), IC41 (Intercell), E-1 (Innogenetics)

Patients receiving hepatitis C medications are typically giveninterferon together with another active agent. Thus methods of treatmentand pharmaceutical combinations in which a compound of the invention isprovided together with an interferon, such as pegylated interferon alfa2a, as the additional active agents are included as embodiments.Similarly methods and pharmaceutical combinations in which ribavirin isan additional active agent are provided herein.

Methods of inhibiting HCV replication in vivo comprising providing acompound or pharmaceutically acceptable salt of Formula (I) to a patientinfected with HCV, a concentration of the compound or salt of Formula(I) sufficient to inhibit HCV replicon replication in vitro are includedherein. In this instance the concentration includes an in vivoconcentration, such as a blood or plasma concentration. Theconcentration of compound sufficient to inhibit HCV replicon replicationin vitro may be determined from an assay of replicon replication such asthe assay provided in Example 11, herein.

Methods of treatment include providing certain dosage amounts of acompound or pharmaceutically acceptable salt of Formula (I) to apatient. Dosage levels of each active agent of from about 0.1 mg toabout 140 mg per kilogram of body weight per day are useful in thetreatment of the above-indicated conditions (about 0.5 mg to about 7 gper patient per day). The amount of active ingredient that may becombined with the carrier materials to produce a single unit dosage formwill vary depending upon the patient treated and the particular mode ofadministration. In certain embodiments about 0.1 mg to about 2000 mg,from about 10 mg to about 1500 mg, from about 100 mg to about 1000 mg,from about 200 mg to about 800 mg, or from about 300 to about 600 mg ofa compound of Formula (I) and optionally from about 0.1 mg to about 2000mg, from about 10 mg to about 1500 mg, from about 100 mg to about 1000mg, from about 200 mg to about 800 mg, or from about 300 to about 600 mgof a compound of an additional active agent, for example an NS3 proteaseinhibitor are provided daily to a patient. It is preferred that eachunit dosage form contains less than 1200 mg of active agent in total.Frequency of dosage may also vary depending on the compound used and theparticular disease treated. However, for treatment of most infectiousdisorders, a dosage regimen of 4 times daily or less is preferred and adosage regimen of 1 or 2 times daily is particularly preferred.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease in the patient undergoing therapy.

Packaged Formulations

Methods comprising providing a compound or salt of Formula (I) in acontainer together with instructions for using the compound to treat apatient suffering from Hepatitis C infection are included herein.

Packaged pharmaceutical compositions/combinations are also includedherein. Such packaged combinations include a compound of Formula (I) ina container together with instructions for using the combination totreat or prevent a viral infection, such as a hepatitis C infection, ina patient.

The packaged pharmaceutical composition/combination may include one ormore additional active agents. In certain embodiments the additionalactive agent is an NS3 protease inhibitor or NS5a inhibitor.

The packaged pharmaceutical combination may include a compound orpharmaceutically acceptable salt of Formula (I) and the additionalactive agent provided simultaneously in a single dosage form,concomitantly in separate dosage forms, or provided in separate dosageforms for administration separated by some amount of time that is withinthe time in which both the compound of Formula (I) and the additionalactive agent are within the bloodstream of the patient.

The packaged pharmaceutical combination may include a compound orpharmaceutically acceptable salt of Formula (I) provided in a containerwith an additional active agent provided in the same or separatecontainer, with instructions for using the combination to treat an HCVinfection in a patient.

EXAMPLES Abbreviations

-   -   Ac₂O Acetic anhydride    -   AcOD Acetic Acid, deuterated    -   BuOH Butanol    -   DCM Dichloromethane    -   EtOAc Ethyl Acetate    -   MTBE Methyl tert-butyl ether    -   PDC Pyridinium Dichromate    -   THF Tetrahydrofuran    -   ^(t)BuMgCl tert-Butyl Magnesium Chloride

Example 1 Preparation of (S)-Isopropyl2-(((S)-(((2R,3R,4R,5R)-5-(5-Deutero-2,4-Dioxo-3,4-Dihydropyrimidin-1(2H)-yl)-3,4-Dihydroxy-4-Methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate(Prodrug of 2′-Methyl-5-Deutero-Uridine)

Tetrabenzoylsugar (1, 2.44 g) and 5-Deutero-Uracil (2, 1.0 g) werereacted following the literature procedure described in Harry-Okuru etal. (J. Org. Chem. (1997) 62: 1754), followed by debenzylation usingNaOMe/MeOH to give 2′-Methyl-S-D-Uridine (3, 0.8 g). Compound 3 (0.7 g)was converted to its phosphoramidate derivative 5 (0.63 g) followingliterature procedure described by Ross et al.

Example 2 Preparation of (S)-Isopropyl2-(((R)-(((2S,3R,4R,5R)-5-(2,4-Dioxo-3,4-Dihydropyrimidin-1(2H)-yl)-3,4-Dihydroxy-4-Methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate(Prodrug of 2′-Methyl-5′,5′-DiDeutero-Uridine)

Compound 6 was prepared from 2′-Me-Uridine (A) following procedurereported by Corey et al (J. Org. Chem. (1984) 49: 47350 with somemodifications.

Lithium chloride (1.76 g) was stirred with NaBD₄ (1.58 g) in EtOD for 1h. Compound 6 (2.97 g) was added to this solution and stirred at roomtemperature for 3 h and quenched with acetic acid-d, diluted with ethylacetate, washed with brine and evaporated to dryness. The residue waspurified by chromatography over silica gel to give 5′-dideuteratedcompound 7 (2.1 g). Compound 7 (2.1 g) was treated with trifluoroaceticacid in presence of water to give the dideuterated nucleoside 8 (1.52g). Compound 8 (1.0 g) was converted to its phosphoramidate derivative 9(0.78 g) following literature procedure described in Ross et al. (J.Org. Chem. (2011) 76: 8311).

Example 3 Preparation of (2S)-Isopropyl2-(((((2S,3R,4R,5R)-1-(6-Deutero-5-(trideuteromethyl)pyrimidine-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate(Compound 16)

Tetrabenzoyl sugar 1 and tetradeutero thymine 10 (2.1 equivalents;prepared according to the procedure in Heterocycles (2005) 66:361) werereacted following the literature procedure described in Harry-Okuru etal. (J. Org. Chem. (1997) 62: 1754), followed by debenzylation usingNaOMe/MeOH to give compound 11.

Excess 2, 2-Dimethyl propane is added to compound 11 in acetone. Theresulting mixture was cooled in an ice bath for 30 min, thenp-Toluenesulfonic acid (1.3 equivalents) was added and the reactionmixture was stirred at room temperature for 24 hrs. After completion ofthe reaction (monitored by HPLC), the reaction mixture is cooled in anice bath for 30 minutes and neutralized using cold aqueous potassiumcarbonate. The solvent is removed under reduced pressure until dryness.THF is added to the residue and solids are removed by filtration. Thefiltrate is co-evaporated with silica gel and purified by chromatographyover silica to give compound 12.

To acetonide 12 in CH₂Cl₂ is added PDC (2.0 equivalents) at roomtemperature followed by Ac₂O (10 equivalents) and tBuOH (20equivalents). The reaction temperature is maintained below 35° C. duringaddition of reagents and then stirred at room temperature for 5 h.

The reaction mixture is poured in to aqueous and the organic layer iswashed with aqueous CuSO₄. Activated charcoal and silica gel are addedto the organic layer and stirred for 30 min and filtered. The filtrateis evaporated and residue purified by chromatography over silica gel togive compound 13.

NaBD₄ (2 equivalents) is added in portions (3-4 portions) to cold (−5°C.) EtOD (99% D) in a flask. Acetonide ester 13 (35 g, 90.10 mmol) wasadded in portions and the resulting reaction mixture was stirred at RTfor 3 hrs, and then heated at 80° C. for 2 days (NMR used to checkgreater than 95% Uridine-5D incorporation). Additional EtOD or D₂O canbe added to increase the deuterium incorporation.

After completion of the reaction, half the solvent is removed underreduced pressure, reaction mixture was cooled in ice bath, AcOD (10equivalents) is added and resulting mixture is stirred for 15-20 minEtOAc and brine are added, organic layer is separated and the aqueouslayer was again extracted with EtOAc followed by THF. The combinedorganic layer was concentrated, residue dissolved in 10% MeOH and CHCl₃,filtered, concentrated and purified by chromatography over silica gel togive deuterated acetonide 14.

Deuterated acetonide 14 is added to cold (˜5° C.) 4N HCl and stirred atroom temperature for 3 h. The solvent was evaporated to dryness and tothe residue was added water and stirred. The suspension was cooled to 5°C., stirred for 1 h and the precipitate was collected by filtration. Thesolid was washed with cold water and dried to afford the deuteratednucleoside 15.

Nucleoside 15 in THF was cooled to −5° C. tBuMgCl (3 equivalents) isadded and stirred for 30 minutes at the same temperature. The reactionmixture is stirred for another 30 minutes at r.t. then cooled again to−5° C. and a solution of 4 in THF (2 equivalents) was added slowly andthen the reaction mixture was stirred at r.t. for 24 h. The reactionmixture was cooled to −5° C. and cold 2N HCl was added, stirred for 10min, and then saturated aqueous NaHCO₃ solution was added followed byaddition of solid NaCl. The mixture was stirred for 1 h and the organiclayer was separated. The aqueous layer was extracted with THF. Allorganic layers are combined and evaporated to dryness. The residue waspurified by chromatography over silica gel to afford the title compound(16).

Example 4 Preparation of (S)-isopropyl2-(((S)-(perfluorophenoxy)(phenoxy) phosphoryl)amino)propanoate(Reactant 4)

The preparation of 4 has previously been reported.

L-analine isopropyl ester HCl salt (160 g, 0.95 mol) is charged in a 5 Lfour-necked flask equipped with mechanical stirrer, thermometer anddropping funnel. To the flask, dichloromethane (1 L) is added and thesuspension is cooled to −70° C., followed by addition of triethylamine(200 g, 276 mL, 1.98 mol) over 45 minutes. To the mixture is added asolution of phenyl dichlorophosphate (200 g, 0.95 mol) indichloromethane (1 L) over 2.5 hours. The reaction mixture is stirred atthis temperature for additional 90 minutes and then allowed to warm upto 0° C. over a period of 2 hr and stirred for 2 hr at 0° C. To themixture a solution of 2,3,5,6-pentafluoro phenol (174.4 g, 0.95 mol) in400 mL dichloromethane and a solution of triethylamine (105.4 g, 1.04mol) in 200 mL dichloromethane are added dropwise simultaneously over aperiod of 1.2 hr. The mixture is warmed to room temperature and stirredovernight.

The solid, triethylamine HCl salt, is filtered off and the cake iswashed with dichloromethane (3×150 mL). The filtrate is concentratedunder reduced pressure and the residue triturated with MTBE (3.0 Liter).The white solid is removed by filtration. The cake is washed with MTBE(3×150 mL). The filtrate is concentrated and the resulting crude solidtriturated with 20% ethyl acetate in hexane (2.0 L). The solid iscollected by filtration and washed with 10% NaHCO₃ until aqueous phasereached pH=7, the solid is then washed with water and dried in a vacuumoven (55° C.) for 28 hr. The dried solid was mixed with 500 mLHexane-Toluene (5:1) and stirred for 1 hr. The solid was collected byfiltration and washed with hexane-toluene (5:1, 2×80 mL) to afford pureone isomer. The solid was dried to give compound 4.

Example 5 Preparation of1-((2R,3R,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)-3-methyltetrahydrofuran-2-yl)-4-alkoxypyrimidin-2(1H)-one

R═OMe, compound 17. Compounds 17 and 18 are prepared by methodswell-known in the chemical literature.17(9): 1236)¹H NMR (CD₃OD): δ 1.00(s, 3H), 3.66 (m, 2H), 3.88 (s, 3H), 3.91 (m, 2H), 5.96 (s, 1H), 5.97(d, 1H, J=7.2 Hz), 8.36 (d, 1H, J=7.2 Hz), LCMS: [M+H]⁺ 273.

R═OEt, compound 18. ¹H NMR (CD₃OD): δ 1.00 (s, 3H), 1.38 (t, 3H, J=7.1Hz), 3.84 (m, 2H), 3.87 (m, 2H), 4.39 (q, 2H, J=7.1 Hz), 6.06 (d, 1H,J=7.4 Hz), 6.08 (s, 1H), 8.47 (d, 1H, J=7.1 Hz), LCMS: [M+H]⁺ 287

Example 6 Synthesis of (S)-isopropyl2-(((S)-phenoxy((2,4,5-trichlorophenyl)thio)phosphoryl)amino)propanoate(Compound 24)

Phenoxydichlorophosphate (22, 12.58 g) is added to a cold (−50° C.)solution of L-Alanine isopropyl ester (21, 10 g) in dichloromethane(DCM, 100 mL), followed by the addition of triethylamine (18.3 mL) inDCM (36 mL) maintained at a temperature below −40° C. The reaction waswarmed to room temperature slowly and stirred for 2 h and again cooledto −50° C. A solution of trichlorothiophenol (23, 12.74 g) in DCM (20mL) containing triethylamine (9.1 mL). The reaction was warmed to roomtemperature and stirred for 15 h.

The reaction mixture was washed with water (˜300 mL) followed bysaturated NaHCO₃ aq (˜300 mL) and the organic layer was collected, driedover Na₂SO₄ and evaporated to dryness. The crude material was passedshort SiO₂ column (CH₂Cl₂/EtOAc=0/1˜1/4) and product collected afterevaporation. The product was dissolved in 100 mL of 2.5% EtOAc inheptane mixture and the solution seeded with compound 24 (˜10 mg) andstirred for 1 h at r.t. The precipitate was collected by filtration andsolid was washed with a small amount of above solvent and dried toafford single isomer 24, 5.2 g (18%).

Trichlorothiophenol (3) may be replaced by other groups such asnitrothiophenol, bromothiophenol, N-hyrdoxysuccinamide,N-hydroxyphthalimide, nitrohydroxypyridine.

Example 7 Alternate Method for Preparing (2S)-isopropyl2-(((((2S,3R,4R,5R)-1-(6-Deutero-5-(trideuteromethyl)pyrimidine-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate(Compound 16)

A suspension of 14 in THF is cooled to −20° C. and ^(t)BuMgCl (3.2equivalents) is added slowly below −20° C. The reaction mixture iswarmed to room temperature slowly (˜2 h) and stirred for 2 h and thencooled again to −10° C. Phosphorous reagent, compound 24 (2.1equivalents), is added and the reaction mixture is warmed to roomtemperature and stirred for 15 h. The reaction mixture is cooled to 0°C. and 2N aq, HCl is added (solution pH ˜2) and stirred for 30 min at 0°C. Then aq NaHCO₃ is added (pH ˜8) followed by NaCl and stirred for 30min. The organic layer is separated, dried and evaporated. Crudematerial is purified by SiO₂ column to afford 16.

Example 8 Alternate Method for Preparing Compound 16

Compound 25 is prepared by the method described in Example 6 forcompound 24. Compound 25 is then reacted with nucleoside 14 by themethod described in Example 6 to give compound 16.

Example 9 Synthesis of (S)-Isopropyl2-(((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphorothioyl)amino)propanoate30 (Comparative Example)

A solution of phenol (15 g) in dichloromethane (DCM) was added to a cold(−78° C.) solution of phosphorothioyl trichloride 26 (16.13 mL) in DCMfollowed by addition of triethylamine (TEA, 22 mL). After the additionwas complete the solution was warmed to room temperature and stirredovernight (˜16 h). DCM was evaporated and the residue triturated bymethyl tert-butylether (MTBE). The solid (triethylamine hydrochloride)was filtered off and the filtrate evaporated to dryness. The residue,O-phenylphosphorodichloridothioate 27, was used without purification inthe next step.

O-Phenylphosphorodichloridothioate 27 from above step was dissolved inDCM, cooled to −78° C., TEA (40 mL) was added followed by a solution ofL-alanine isopropyl ester (28 g) in DCM while maintaining the reactiontemperature below −60° C. The reaction was stirred or 30 min and thenwarmed to room temperature and stirred overnight (˜16 h). The solventwas evaporated and residue triturated with MTBE and solid triethylaminehydrochloride removed by filtration. The filtrate was evaporated todryness and the residue purified by chromatography over silica gel(eluted with 0-1.5% ethylacetate/hexane). The pure fraction werecollected and evaporated to obtain 20 g of thiophosphoryl chloride 28.

N-Methylimidazole (12 mL) was added to a solution of 2′-C-methyluridine29 (6.34 g) in acetonitrile and cooled to −10° C. A solution of compound28 (7.9 g) in acetonitrile was then added. The reaction mixture wasstirred at 0° C. for 1 h and then at room temperature overnight (˜16 h).The solvent was evaporated and the residue purified by chromatographyover silica gel (eluted with 0-2.5% methanol/DCM). The pure fractionswere mixed and evaporated to dryness and the residue crystallized fromMTBE. The solid obtained was suspended in MTBE and refluxed for 2 hcooled and filtered. The solid was washed with MTBE and dried to give0.850 g of compound 30 (3b(ii)-Sp from application WO 2012/040127).About 3.5% of the other isomer Rp is present.

3b(ii)-Sp: ¹H-NMR (CD₃OD, 400 MHz): δ 7.76 (d, J=8.4 Hz, 1H), 7.34 (t,J=8.4 Hz, 2H), 7.27 (d, J=8.8 Hz, 2H), 7.18 (t, J=7.6 Hz, 1H), 5.96 (s,1H), 5.57 (d, J=8.4 Hz, 1H), 4.98 (m, 1H), 4.5 (m, 1H), 4.3 (m, 1H), 4.1(m, 2H), 3.81 (d, J=9.2 Hz, 1H), 1.37 (d, J=6.8 Hz, 3H), 1.23, 1.22 (2d,J=6.8 Hz and 6 Hz, 6H), 1.15 (s, 3H); ³¹P-NMR (CD₃OD, 162 MHz): δ 68.42(96.5%) and 68.21 (3.5%); ESI-LCMS: m/z=544 [M+1].

Example 10 Preparation of (S)-isopropyl2-(((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,4-dihydroxy-4-trideuteromethyltetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino)propanoate

Into a flask charged with magnesium turnings (0.25 g) and THF (1 mL) isadded CD₃I (0.65 mL) dropwise over 30 min at rt under Ar with gentlestirring. The reaction mixture is stirred at rt for an additional 1 h.The resulting cloudy mixture is cooled to −78° C., and then 31 (1.0 g)in THF (1.5 mL) is added. The reaction mixture is allowed to warm to rtover 2 h and then aq NH₄Cl (10 mL) is added to quench the reaction. Themixture is extracted with EtOAc (50 mL). After washing the organic layerwith brine (10 mL) and drying over anhydrous Na₂SO₄, the solvent isremoved under reduced pressure to give compound 32 as a yellow syrup.

Compound 32 (0.878 g) is dissolved in CH₂Cl₂ (25 mL) and treated withBzCl (0.51 mL) in the presence of triethylamine (3.1 mL) and DMAP (53mg) overnight. Volatiles are removed by under reduced pressure, theresidue is treated with water, and the product is extracted with EtOAc.After washing the organic layer with aq NaHCO₃ and brine, followed bydrying over anhydrous Na₂SO₄, the solvent is removed under reducedpressure to give the crude product. This material is purified by columnchromatography on silica gel (hexanes/EtOAc 4:1 v/v as eluent) to give33 as yellow syrup.

Uracil (0.394 g) in acetonitrile (15 mL) is treated with BSA(N,O-bis(trimethylsilyl)acetamide, 1.72 mL) at 70° C. under Ar for 1 h.The clear solution is cooled to 0° C., then compound 33 (0.53 g) inacetonitrile (5 mL) followed by SnCl₄ (0.413 mL) is added. The reactionmixture is stirred at 70° C. under Ar for 3 d and quenched by additionof a saturated aq solution of NaHCO₃ at 0° C. CH₂Cl₂ is added and thenthe mixture is filtered through a Celite pad to remove solids. Thefiltrate is dried over anhydrous Na₂SO₄ and evaporated under reducedpressure to give 34 as a foam.

Compound 34 (0.58 g) is dissolved in MeOH/THF (5 mL/2 mL) and treatedwith NaOMe/MeOH (30%, 0.2 mL) at rt overnight. Volatiles are removedunder reduced pressure. The remaining residue is treated with HCl (10%aq, 0.4 mL) and then purified by column chromatography on silica gel (5%MeOH in CH₂Cl₂ as eluent) to give 35 as a syrup.

Compound 35 (0.418 g) is dissolved in MeOH (15 mL) and treated with H₂(˜1 atm, balloon) in the presence of Pd(OH)₂ on carbon (20% wet, 50 mg)overnight. After filtration and evaporation of the filtrate, theremaining residue was purified by column chromatography on silica gel(15% MeOH in CH₂Cl₂ as eluent) to give nucleoside 36 as a colorlesssolid. ¹H NMR (400 MHz, CD₃OD, 300 K): δ 3.78 (dd, J=12.5 Hz, 2.6 Hz,1H), 3.84 (d, J=9.2 Hz, 1H), 3.92 (d of app t, J=9.2 Hz, 2.4 Hz, 1H),3.98 (dd, J=12.5 Hz, 2.2 Hz, 1H), 5.67 (d, J=8.1 Hz, 1H), 5.96 (s, 1H),8.14 (d, J=8.1 Hz, 1H); ¹³C NMR (100 MHz, CD₃OD, 300 K): δ 60.5, 73.4,79.9, 83.9, 93.1, 102.3, 142.5, 152.5, 166.0 (CD₃ not observed); LC-MS:262 amu (M+1).

Nucleoside 36 was converted to the phosphoramidate derivative 37 in amanner analogous to that described in Example 1. ¹H NMR (400 MHz, CD₃OD,300 K): δ 1.21 (2×d, J=6.3 Hz, 6H), 1.35 (dd, J=7.2 Hz, J_(H,P)=0.9 Hz,3H), 3.79 (d, J=9.2 Hz, 1H), 3.91 (dq, J_(H,P)=10.0 Hz, J=7.2 Hz, 1H),4.08 (m, 1H), 4.37 (ddd, J=11.8 Hz, J_(H,P)=5.9 Hz, J=3.7 Hz, 1H), 4.50(ddd, J=11.8 Hz, J_(H,P)=5.9 Hz, J=2.0 Hz, 1H), 4.96 (septet, J=6.3 Hz,1H), 5.60 (d, J=8.1 Hz, 1H), 5.96 (s, 1H), 7.20 (m, 1H), 7.26 (m, 2H),7.37 (m, 2H), 7.67 (d, J=8.1 Hz, 1H); ³¹P NMR (162 MHz, CD₃OD, 300 K): δ3.8; LC-MS: 531 amu (M+1).

Example 11 Preparation of (S)-isopropyl2(((S)-(((2R,3R,4R,5R)-3,4-dihydroxy-5-(5-deutero-4-methoxy-2-oxopyrimidin-1(2H)-yl)-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate(Compound 39)

Compound 38 is prepared in a manner analogous to that described in Kanget al. (Chem. Res. Toxicol. (2004) 17: 1236). Nucleoside 38 is convertedto the phosphoramidate derivative 39 in a manner analogous to thatdescribed in Example 2. ¹H NMR (400 MHz, CD₃OD, 300 K): δ 1.11 (s, 3H),1.23 (d, J=6.3 Hz, 6H), 1.37 (dd, J=7.1 Hz, 0.8 Hz, 3H), 3.79 (d, J=9.3Hz, 1H), 3.90-3.99 (overlapping s and m, 4H), 4.14 (dd, J=9.3 Hz, 2.3Hz, 1H), 4.97 (septet, J=6.3 Hz, 1H), 6.09 (s, 1H), 7.22 (m, 1H), 7.29(m, 2H), 7.39 (m, 2H), 8.00 (s, 1H); ³¹P NMR (162 MHz, CD₃OD, 300 K): δ3.8; LC-MS: 545 amu (M+1).

Example 12 Preparation of (S)-isopropyl2-(((S)-(((2R,3R,4R,5R)-5-(5-deutero-4-ethoxy-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)dideuteromethoxy)(phenoxy)phosphoryl)amino)propanoate(Compound 41)

Compounds 40 and 41 are prepared using methods analogous to thosedescribed in Example 11. Spectroscopic data for 41: ¹H NMR (400 MHz,CD₃OD, 300 K): δ 1.11 (s, 3H), 1.23 (d, J=6.3 Hz, 6H), 1.38 (overlappingd and t, 6H), 3.79 (d, J=9.3 Hz, 1H), 3.94 (m, 1H), 4.14 (dd, J=9.3 Hz,2.3 Hz, 1H), 4.41 (q, J=7.0 Hz, 2H), 4.97 (septet, J=6.3 Hz, 1H), 6.08(s, 1H), 7.22 (m, 1H), 7.29 (m, 2H), 7.39 (m, 2H), 7.99 (s, 1H); ³¹P NMR(162 MHz, CD₃OD, 300 K): δ 3.8; LC-MS: 559 amu (M+1).

Example 13 Additional Compounds

The following compounds are prepared by the methods given in Examples1-9. In each example R=methoxy or ethoxy.

Example 14 Determination of Anti-HCV Activity and Cytotoxicity

Compounds claimed herein are tested for the ability to inhibit viralreplication of the Hepatitis C replicon in cultured cells in which theHCV replicon construct has been incorporated. The replicon system ispredictive of in vivo anti-HCV activity; compounds that are active inhumans uniformly evidence activity in the replicon assay. In this assayHCV replicon containing cells are treated with different concentrationsof the test compound to ascertain the ability of the test compound tosuppress replication of the HCV replicon.

The Cell Line

The Huh-luc/neo cell line was obtained from ReBLikon GmbH (Mainz,Germany) [Error! Reference source not found.]. This cell line harbors abicistronic genotype-1b/strain Con-1 HCV subgenomic replicon: the firstcistron encodes both luciferase and neomycin phosphotransferase II(NPTII) and the second cistron encodes HCV non-structural proteins NS3through NS5B. Hence, the luciferase activity in the cultured cells wasused as a surrogate marker for the level of HCV replicon RNA. Thereplicon cells were maintained in complete medium [DMEM (LifeTechnologies, Carlsbad, Calif.), 10% fetal bovine serum (FBS),1×non-essential amino acids (Life Technologies, Carlsbad, Calif.), andpenicillin (100 IU/mL), and streptomycin (100 μg/mL)], with addition of0.25 mg/mL G418 (Life Technologies, Carlsbad, Calif.) and were passagedtwice a week.

Huh-Lunet cells were derived from Huh-luc/neo cells (Vrolijik, J. M, etal. “J. Virol. Methods (2003) 110(2): 201-209) by eliminating HCVreplicons with a selective HCV inhibitor. Huh-Lunet cells weremaintained in a complete medium consisting of DMEM, 10% FBS,non-essential amino acids, penicillin, and streptomycin at 37° C. in anatmosphere of 5% CO₂.

Determination of Anti-HCV Activity and Cytotoxicity

Huh-luc/neo cells were seeded in 96-well plates at a density of 8×10³cells per well in 200 μL DMEM supplemented with 10% FBS. One day afterseeding, compounds were prepared as six half-log dilution series in 100%DMSO and added to cells at a 1:200 ratio, achieving DMSO finalconcentration of 0.5% in a total volume of 200 μL. Cell plates wereincubated at 37° C. for 3 days. The anti-HCV activity of ACH-0143422 wasdetermined by quantifying luciferase activity in each well with aBright-Glo Luciferase Assay kit (Promega, Madison, Wis.). Due to thewide dynamic range offered by the luciferase activity, anti-HCV activitycould be expressed as the concentration that caused a reduction ofluciferase activity (relative luminescence units, RLU) by 50% (EC₅₀) incomparison to the untreated controls. EC₅₀ values were calculated with aMicrosoft Excel-based program. Cellular toxicity of the compound wasdetermined by measuring the cell viability in each well with a CellTiter96 AQ_(ueous) One Solution kit (Promega, Madison, Wis.). Theconcentration of compound that caused a reduction of the cell viabilityby 50% (CC₅₀) relative to untreated cells was obtained with a MicrosoftExcel-based program.

Example 15 Determination of Anti HCV Activity Using Transient HCVReplicons Plasmids Encoding Transiently Replicating Chimeric Replicons

This assay utilizes plasmids encoding transiently replicating chimericreplicons carrying the coding region of NS5B from HCV genotype 3a or 4a.The plasmid pFK-I341PI-luc/NS3-3′/ET (ReBLikon GmbH, Germany) (Lohmann,V., et al, (J. Virol. (2003) 77(5): 3007-3019) was used as backbone forthe chimeric replicon constructions. This plasmid encodes a repliconthat carries a luciferase reporter gene driven by the poliovirus IRESand the HCV NS3 to NS5B nonstructural genes from genotype-1b/strain-Con1HCV driven by the EMCV IRES. Three adaptive mutations, E1202G and T1280Iin NS3 and K1846T in NS4B, were introduced into this construct forefficient replicon replication. The NS5B sequences of HCV genotypes 3aand 4a were synthesized by Integrated DNA Technologies, Inc (Coralville,Iowa). NS5B DNA fragments were inserted in-frame intopFK-I₃₄₁PI-luc/NS3-3′/ET.

Synthesis of HCV Replicon RNA

To generate run-off transcripts of HCV replicons, the plasmids encodingvarious HCV replicons were linearized by digestion with ScaI. Afterextraction with phenol-chloroform and ethanol precipitation, theplasmids were used as templates for in vitro T7 transcription reactions(Megascript T7 kit, Ambion, Austin, Tex.). Transcripts were extractedonce with acidic phenol and chloroform. After isopropanol precipitation,RNA was dissolved in RNase-free water and concentrations were determinedby measurement of the optical density at 260 nm.

Transient HCV Replicon Assay

Replicon RNAs were transfected into Huh-Lunet cells by electroporation.In brief, single-cell suspensions of Huh-Lunet cells were prepared at adensity of 10⁷ cells per mL in Cytomix solution supplemented with 2 mMATP and 5 mM glutathione. After mixing 5 μg RNA with 400 μL of the cellsuspension in a Gene Pulser cuvette (0.4 cm gap), electroporation wasimmediately performed at 270V and 950 μF with a Gene Pulser system(Bio-Rad, Hercules, Calif.). Electroporated cells were immediatelydiluted into 10 mL DMEM supplemented with 10% FBS and seeded into96-well plates at a density of 8×10³ cells per well in a final volume of200 μL DMEM supplemented with 10% FBS. One day after seeding, compoundswere prepared as six half-log dilution series in 100% DMSO and added tocells at a 1:200 ratio, achieving DMSO final concentration of 0.5% in atotal volume of 200 μL. Cell plates were incubated at 37° C. for 3 days.The inhibition of HCV replicon replication was quantified by measurementof firefly or Renilla luciferase activity using commercial kits(Bright-Glo Luciferase Assay or Renilla-Glo Luciferase Assay, Promega,Madison, Wis.). Anti-HCV activity was expressed as the concentrationthat reduced luciferase activity by 50% (EC₅₀) compared to the untreatedcontrols. EC₅₀ values were calculated with a Microsoft Excel-basedprogram.

Certain compounds of Formula (I) exhibit an EC₅₀ of less than 0.1micromolar when evaluated in the replicon assay using thegenotype-1b/strain Con-1 HCV subgenomic replicon. Furthermore certaincompounds of Formula (I) exhibit a selectivity index (CC₅₀/EC₅₀ ofgreater than 100 and in some instances greater than 500. Furthermorecertain compounds of Formula (I) exhibit an EC₅₀ that is significantlyless than the EC₅₀ exhibited by comparative compound 30 (3b(ii)-Sp) whenevaluated in the replicon assay using the genotype-1b/strain Con-1 HCVsubgenomic replicon. In some instances a compound of Formula (I)exhibits and EC₅₀ that is at least 5-fold less than the EC₅₀ ofcomparative compound 30. Furthermore certain compounds of Formula (I)exhibit an improved CC₅₀/EC₅₀ selectivity index relative to comparativecompound 30. In some instances the selectivity index for a compound ofFormula (I) is more than 5-fold greater than the selectivity index forcomparative compound 30. Furthermore certain compounds of Formula (I)exhibit improved potency (lower EC₅₀) over comparative compound 30 whenevaluated in the replicon assay against genotypes 3a and 4a replicons.

Example 16 Determination of Nucleoside Concentrations in HumanHepatocytes

This assay is used to determine the concentration free nucleoside inmedia and hepatocyte cell extract for nucleoside prodrugs incubated withfresh liver hepatocytes. LC MS can also be used to detect the level ofnucleoside monophosphate. A higher concentration of free nucleoside isdirectly related to a decrease in nucleoside prodrug and the loss ofdrug activity.

Cells

Fresh human liver hepatocytes were received plated in a 12-well and6-well format. Upon receipt, shipping media was removed immediately andreplaced with 1 mL or 2 mL pre-warmed culture medium. Cells were platedate a density of 0.67 million cells per well in the 12 well format and1.7 million cells per well in the 6 well format. Supplemented modifiedChee's media (Xenotec, LLC, catalogue no. K2300) was used as the culturemedia. Cells were acclimated overnight at 37° C. with 5% CO₂ atmosphere.

Assay

Media was aspirated from 12- and 6-well plates and replaced with 1 mL or2 mL respectively of fresh media containing either 20 μM deuteratedprodrug or 20 μM ACH-undeuterated prodrug or solvent control (0.05%DMSO). Samples incubated at 37° C. in 5% CO₂ atmosphere were induplicate for deuterated prodrug and in singlet undeuterated prodrug ineach well format. Stability of compound in absence of cells was alsoconducted

At 24 hrs, media was removed and frozen. Cells were washed twice withcold PBS. 70% cold Methanol (0.75 mL or 1.5 mL for 12- and 6-wellrespectively) containing internal standard, an non-deuterated prodrugwith known anti-HCV efficacy was added to each well and cells weregently removed from the plate by scraping. The recovered cells suspendedin the organic solution were aspirated into a vial and frozen at −80° C.

Extraction and LC-MS/MS Analysis of Hepatocyte Cells

Cell solutions extracted overnight at −80° C. in 70% Methanol wereremoved from the freezer, defrosted and vortexed. Tubes were centrifugedat 3000 rpm for 15 minutes at 4° C. Supernatants were removed andanalyzed by LC-MS/MS.

Six concentrations of deuterated prodrug, non-deuterated prodrug, freenucleoside of the deuterated prodrug, or free nucleoside on thenon-deuterated prodrug were prepared by 3-fold serial dilution in DMSO.Aliquots of the compounds at the specified concentrations were spikedinto 70% methanol containing internal standard. 2 concentrations werealso spiked into cells solutions from the experiment incubated in theabsence of compound. Samples were frozen at −80° C. overnight, thendefrosted and vortexed. Samples were centrifuged at 3000 rpm for 15minutes. Supernatants were removed and analyzed by LC-MS/MS. Thecalibration concentrations were 5, 1.67, 0.556, 0.185, 0.0617 and 0.0206μM.

The analytes were quantified using linear regression of calibrationstandard values with instrument response. The acceptance criteria usedand calibration standard concentrations was ±30% of nominalconcentration. Calibration standards that did not meet the specifiedcriteria were not used in the calibration curve. Sample values wereaccepted when at least 66% of the standard concentrations during the runwere within 30% of nominal. The “r” value required for acceptance of therun was >0.98. Cell samples were analyzed without internal standard dueto only 81% extraction efficiency of internal standard from cells whilecalibration was conducted without cells, this gave more accuratedetermination of concentrations.

Extraction and LC-MS/MS Analysis of Hepatocyte Media

Hepatocyte media incubates were removed from the freezer, defrosted andvortexed. 2 parts hepatocyte media incubate to 1 part Acetonitrilecontaining internal standard were mixed and then centrifuged at 3000 rpmfor 15 minutes at 4° C. Supernatants were removed and analyzed byLC-MS/MS.

Six concentrations of deuterated prodrug, non-deuterated prodrug, freenucleoside of the deuterated prodrug, or free nucleoside of thenon-deuterated prodrug were prepared by 3-fold serial dilution in DMSO.Aliquots of the compounds were spiked into fresh hepatocyte media toafford 5, 1.67, 0.556, 0.185, 0.0617 and 0.0206 μM concentrations. 2parts calibration media were mixed with 1 part Acetonitrile containinginternal standard samples were centrifuged at 3000 rpm for 15 minutes at4° C. Supernatants were removed and analyzed by LC-MS/MS.

Analyte concentrations in the samples were quantified using linearregression of calibration standard values with instrument response. Theacceptance criteria used and calibration standard concentrations was±30% of nominal concentration. Calibration standards that did not meetthe specified criteria were not used in the calibration curve. Samplevalues were accepted when at least 66% of the standard concentrationsduring the run were within 30% of nominal. The “r” value required foracceptance of the run was >0.98.

Certain compounds of this disclosure had increased concentrations (morethat 1.5-fold) in the media and hepatocyte cell extract of the freenucleoside for the non-deuterated prodrug relative to the concentrationof the free nucleoside for the otherwise identical deuterated nucleosideprodrug.

1. A compound of the Formula (I)

or a pharmaceutically acceptable salt thereof, wherein: Y is NH or O; R₁is hydroxyl, fluoro, or —OCD₃; R₂ is hydrogen or deuterium; or R₂ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, or C₂-C₆alkynyl; each of which isoptionally deuterated and optionally substituted; or R₁ and R₂ arejoined to form a 3- to 6-membered cycloalkyl ring or a 3- to 6-memberedheterocycloalkyl ring containing one heteroatom selected from N, O, andS, each of which is optionally substituted; R₃ is hydrogen, deuterium,halogen, or —N₃; or R₃ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, (C₃-C₆cycloalkyl)C₀-C₄carbhydryl, (4- to 6-memberedheterocycloalkyl)C₀-C₄carbhydryl, (aryl)C₀-C₄carbhydryl, or(heteroaryl)C₀-C₄carbhydryl, each or which is optionally deuterated andoptionally substituted; and R₃′ is hydroxyl; or R₃ and R₃′ are takentogether to form a 3- to 6-membered ring optionally containing oneheteroatom selected from N, O, and S, which is optionally substitutedwith one or more substituents independently selected from halogen,hydroxyl, C₁-C₂alkyl, and C₁-C₂alkoxy; R₄ is hydrogen, deuterium,halogen, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy; or R₄ is C₁-C₆alkyl,allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or C₁-C₆alkoxy, each of which isoptionally deuterated and optionally substituted; R₅ is hydrogen,deuterium, or halogen; or R₅ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, or C₁-C₆alkoxy, each of which is optionally deuterated andoptionally substituted; or R₄ and R₅ are taken together to form a 3- to6-membered ring optionally containing one heteroatom selected from N, O,and S, which ring is optionally substituted with one or moresubstituents independently selected from halogen, hydroxyl, C₁-C₂alkyl,and C₁-C₂alkoxy; R₆ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl,(aryl)C₀-C₂alkyl, or 5- to 6-membered monocyclic heteroaryl containing 1to 3 heteroatoms independently chosen from N, O, and S, or 8- to10-membered bicyclic heteroaryl containing 1 to 4 heteroatomsindependently chosen from N, O, and S; each of which R₆ is optionallysubstituted; R₇ is hydrogen, halogen, C₁-C₂haloalkyl, orC₁-C₂haloalkoxy; or R₇ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆alkoxy, (C₃-C₆cycloalkyl)C₀-C₄alkyl, or(aryl)C₀-C₂alkyl; each of which is optionally substituted; R₈ ishydrogen, halogen, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy; or R₈ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or C₁-C₆alkoxy, each ofwhich is optionally substituted; or R₇ and R₈ are taken together to forma 3- to 6-membered cycloalkyl ring or 3- to 6-membered heterocycloalkylring containing one heteroatom chosen from N, O, and S; each of which isoptionally substituted; R₉ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, (C₃-C₇cycloalkyl)C₀-C₄carbhydryl, (aryl)C₀-C₄carbhydryl,(3- to 6-membered hetero cycloalkyl)C₀-C₄carbhydryl, or(heteroaryl)C₀-C₄carbhydryl, each of which is optionally substituted; B₁is a base selected from

R₁₀ and R₁₁ are independently hydrogen and deuterium; R₁₂, R₁₃, and R₁₃′are independently hydrogen, deuterium, methyl, and —CD₃; R₁₄ ishydrogen, deuterium, hydroxyl, amino, C₁-C₄alkoxy, deuteratedC₁-C₄alkoxy, C₁-C₄alkylester, or mono- or di-C₁-C₄alkylcarbamate;wherein each position represented by D has a deuterium enrichment of atleast 50%; and one or both of R₄ and R₅ is deuterium with a deuteriumenrichment of at least 50%; and one or more of R₁, R₂, R₁₀, R₁₁, R₁₂,R₁₃, R₁₃′, and R₁₄ is optionally deuterium with a deuterium enrichmentof at least 50% or a deuterated substituent with at least one positionof the substituent having a deuterium enrichment of at least 50%. 2-5.(canceled)
 6. The compound or salt of claim 1, wherein each positionrepresented by D has a deuterium enrichment of at least 90%; and atleast one of R₄ and R₅ is deuterium with a deuterium enrichment of atleast 90% or a deuterated substituent with at least one position of thesubstituent having a deuterium enrichment of at least 90%.
 7. A compoundor salt of claim 2, where Y is NH.
 8. A compound or salt of claim 7,where R₁ is hydroxyl, fluoro, or —OCD₃; R₂ is hydrogen or deuterium; orR₂ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl, or C₂-C₆alkynyl; each of whichis optionally deuterated and optionally substituted.
 9. (canceled)
 10. Acompound or salt of claim 8, wherein R₁ is hydroxyl or fluoro; and R₂ ismethyl or —CD₃. 11-13. (canceled)
 14. A compound or salt of claim 10,wherein R₃ is hydrogen, deuterium, halogen, or —N₃ and R₃′ is hydroxyl.15-18. (canceled)
 19. A compound or salt of claim 14, wherein both R₄and R₅ are both deuterium. 20-24. (canceled)
 25. A compound or salt ofclaim 19, wherein R₇ and R₈ are independently chosen from hydrogen,halogen, C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.26-27. (canceled)
 28. A compound or salt of claim 25, wherein R₉ isC₁-C₆alkyl.
 29. A compound or salt of claim 28, wherein B₁ is apyrimidine base chosen from


30. (canceled)
 31. A compound or salt of claim 6, of Formula (II)

or a pharmaceutically acceptable salt thereof, wherein: R₁ is hydroxyl,fluoro, or —OCD₃; R₂ is hydrogen, —CH₃, or —CD₃; or R₁ and R₂ are joinedto form a cyclopropyl; R₃ is hydrogen or —N₃; R₄ and R₅ areindependently hydrogen, deuterium, methyl, or —CD₃; R₆ is phenyl,pyridyl, naphthyl, or indolyl, each of which is optionally substitutedwith one or more substituents independently chosen from halogen,hydroxyl, amino, cyano, —CHO, —COOH, —CONH₂, C₁-C₆alkyl, C₂-C₆alkenyl,C₁-C₆alkoxy, C₂-C₆alkanoyl, C₁-C₆alkylester, (mono- anddi-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy; R₇is hydrogen, halogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₂haloalkyl,C₁-C₂haloalkoxy, (C₃-C₆cycloalkyl)C₀-C₄alkyl, or (aryl)C₀-C₂alkyl; R₈ ishydrogen, halogen, C₁-C₆alkyl, C₁-C₆alkoxy, C₁-C₂haloalkyl, orC₁-C₂haloalkoxy; or R₇ and R₈ are taken together to form a 3- to6-membered cycloalkyl ring; and R₉ is C₁-C₆alkyl,(C₃-C₇cycloalkyl)C₀-C₄alkyl, or (aryl)C₀-C₄alkyl, each of which isoptionally substituted with one or more substituents independentlychosen from halogen, hydroxyl, amino, cyano, —CHO, —COOH, —CONH₂,C₁-C₆alkyl, C₂-C₆alkenyl, C₁-C₆alkoxy, C₂-C₆alkanoyl, C₁-C₆alkylester,(mono- and di-C₁-C₆alkylamino)C₀-C₂alkyl, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy; B₁ is a base selected from

where R₁₀ and R₁₁ are independently hydrogen and deuterium; R₁₂ and R₁₃are independently hydrogen, deuterium, and methyl; R₁₄ is hydrogen,deuterium, hydroxyl, amino, C₁-C₄alkoxy, deuterated C₁-C₄alkoxy,C₁-C₄alkylester, or mono- or di-C₁-C₄alkylcarbamate. 32-53. (canceled)54. A compound or salt of claim 31 wherein R₃ is hydrogen.
 55. Acompound or salt of claim 54 wherein R₃ is —N₃.
 56. (canceled)
 57. Acompound or salt of claim 55, wherein R₆ is unsubstituted phenyl. 58-59.(canceled)
 60. A compound or salt of claim 57, wherein R₇ is methyl andR₈ is hydrogen.
 61. A compound or salt of claim 60, wherein R₉ isC₁-C₆alkyl. 62-71. (canceled)
 72. A pharmaceutical compositioncomprising a compound or salt claim 6 together with a pharmaceuticallyacceptable carrier. 73-74. (canceled)
 75. The pharmaceutical compositionof claim 72, additionally comprising an NS5a inhibitor and at least oneof sovaprevir and ACH-2684.
 76. A method of treating hepatitis Cinfection in a patient, comprising administering an effective amount ofa compound or salt claim
 6. 77-83. (canceled)
 84. An intermediate of theformula

where Ar is an optionally substituted aryl, heteroaryl, orheterocycloalkyl group; L is O or S; R₆ is C₁-C₆alkyl, allenyl,C₂-C₆alkenyl, C₂-C₆alkynyl, (aryl)C₀-C₂alkyl, or 5- to 6-memberedmonocyclic heteroaryl containing 1 to 3 heteroatoms independently chosenfrom N, O, and S, or 8- to 10-membered bicyclic heteroaryl containing 1to 4 heteroatoms independently chosen from N, O, and S; each of which R₆is optionally substituted; R₇ is hydrogen, halogen, C₁-C₂haloalkyl, orC₁-C₂haloalkoxy; or R₇ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆alkoxy, (C₃-C₆cycloalkyl)C₀-C₄alkyl, or(aryl)C₀-C₂alkyl; each of which is optionally substituted; R₈ ishydrogen, halogen, C₁-C₂haloalkyl, or C₁-C₂haloalkoxy; or R₈ isC₁-C₆alkyl, allenyl, C₂-C₆alkenyl, C₂-C₆alkynyl, or C₁-C₆alkoxy, each ofwhich is optionally substituted; or R₇ and R₈ are taken together to forma 3- to 6-membered cycloalkyl ring or 3- to 6-membered heterocycloalkylring containing one heteroatom chosen from N, O, and S; each of which isoptionally substituted; R₉ is C₁-C₆alkyl, allenyl, C₂-C₆alkenyl,C₂-C₆alkynyl, (C₃-C₇cycloalkyl)C₀-C₄carbhydryl, (aryl)C₀-C₄carbhydryl,(3- to 6-membered hetero cycloalkyl)C₀-C₄carbhydryl, or(heteroaryl)C₀-C₄carbhydryl, each of which is optionally substituted.85-86. (canceled)